Proteins for the Treatment of Epithelial Barrier Function Disorders

ABSTRACT

The disclosure relates to therapeutic proteins and pharmaceutical compositions comprising said proteins, which have utility in treating various human diseases. In particular aspects, the disclosed therapeutic proteins are useful for treating human gastrointestinal inflammatory diseases and gastrointestinal conditions associated with decreased epithelial cell barrier function or integrity. Further, the disclosed therapeutic proteins are useful for treating human inflammatory bowel disease, including inter alia, Crohn&#39;s disease and ulcerative colitis.

This application claims the benefit of U.S. Provisional Application No.62/654,083, filed on Apr. 6, 2018, the content of which is herebyincorporated by reference in its entirety.

FIELD

The present disclosure relates to novel proteins and pharmaceuticalcompositions comprising said proteins that have application, inter alia,in the treatment of gastrointestinal inflammatory diseases andepithelial barrier function disorders. In some embodiments, the proteinsand pharmaceutical compositions described herein have particularapplication in the treatment or prevention of disease states associatedwith abnormally permeable epithelial barriers as well as inflammatorybowel diseases or disorders.

BACKGROUND

Inflammatory bowel disease (IBD) is a diverse disease of unknownetiology resulting in more frequent and bloody bowel movementsaccompanied with histopathological damage to the gastrointestinal mucosa(Zhang et al., 2017, Front Immunol, 8:942). While specific triggers ofdisease remain poorly defined, one proposal of disease progressionsuggests that a breakdown of intestinal barrier function allows bacteriaor bacterial components to translocate into mucosal tissue (Coskun,2014, Front Med (Lausanne), 1:24; Martini et al., 2017, Cell MolGastroenterol Hepatol, 4:33-46). Bacterial translocation results inactivation of inflammatory signaling which triggers additional barrierdisruption, resulting in a cyclic amplification loop of barrierdisruption, bacterial translocation and inflammation. While many currenttherapies target inflammation, the lack of therapies promoting mucosalhealing provides an opportunity for novel therapies promoting epithelialrepair and intestinal barrier integrity.

Expanding upon the hypothesis that bacterial translocation can triggerIBD, more recent studies have demonstrated detrimental changes inintestinal microbiota, or dysbiosis, may promote development of IBD.

Currently, many IBD therapeutics available in the market merely aim totarget and suppress the discussed inflammatory response associated withIBD. While helpful, this narrow therapeutic mode of action disregardsthe important contribution that epithelial barrier integrity plays inthe etiology of the disease.

Thus, there is a great need in the art for the development of a proteintherapeutic, which not only suppresses the immune system's inflammatoryresponse, but that also acts in concert to restore the epithelialbarrier function in an individual. Also, there is an unmet need for theproduction of a protein therapeutic that is stable through themanufacturing and/or processing of the protein therapeutic as well asunder long term storage conditions.

SUMMARY OF THE DISCLOSURE

The present disclosure addresses the important need in the medicalcommunity for a therapeutic which can effectively treat a subjectsuffering from a gastrointestinal disorder such as an inflammatory boweldisease (IBD). In one aspect, novel protein therapeutics are providedwhich can improve and/or maintain epithelial barrier integrity. Theseprotein therapeutics can also reduce inflammation of the intestine ofthe subject and/or decrease symptoms associated with inflammation of theintestine.

The protein therapeutics provided herein are useful in treating thenumerous diseases and/or symptoms that may be associated with decreasedgastrointestinal epithelial cell barrier function or integrity.

In some embodiments, the disclosure provides novel protein therapeuticsderived from the microbiome and methods of utilizing said proteintherapeutics. In a particular embodiment, a protein derived from themicrobiome and comprising an amino acid sequence having at least about70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,or 100%, sequence identity to SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49is provided. In some embodiments, the therapeutic protein does notcomprise an amino acid sequence identical to SEQ ID NO:3 or SEQ IDNO:34. In yet other embodiments, the therapeutic protein comprises anamino acid sequence which is not naturally occurring.

In some embodiments, the disclosure provides novel protein therapeuticsderived from the microbiome and methods of utilizing said proteintherapeutics. In a particular embodiment, a protein derived from themicrobiome and comprising an amino acid sequence having at least about70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,or 100%, sequence identity to SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49 is provided. In someembodiments, the therapeutic protein does not comprise an amino acidsequence identical to SEQ ID NO:3 or SEQ ID NO:34. In yet otherembodiments, the therapeutic protein comprises an amino acid sequencewhich is not naturally occurring.

In some embodiments, the protein comprises the amino acid sequence ofSEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 orSEQ ID NO:49. In other embodiments, the protein comprises the amino acidsequence of SEQ ID NO:3. In still other embodiments, the proteincomprises the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:42.

In some embodiments, the protein comprises an amino acid sequence whichis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical toSEQ ID NO:34, wherein the amino acid sequence has at least 1, 2, 3 or 4amino acid substitutions relative to SEQ ID NO:34 or to SEQ ID NO:36. Inother embodiments, the amino acid sequence has at least 2 and less than3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acidsubstitutions relative to SEQ ID NO:34. In still other embodiments, thetherapeutic protein comprises an amino acid sequence which is notnaturally occurring.

In some embodiments, the protein comprises the amino acid sequence ofSEQ ID NO:34. In other embodiments, X11 is N, R or K; and/or X12 is G orA; and/or X75 is C, S, T, M, V, L, A, or G; and/or X79 is C, S, T, M, V,L, A, or G. In still other embodiments, X11 is N or R; and/or X12 is Gor A; and/or X75 is C, V, L or A; and/or X79 is C, S, V, L or A.

In some embodiments, the protein is about 100 to 200 amino acids, 110 to190 amino acids, 120 to 180 amino acids, 130 to 170 amino acids, 140 to170 amino acids, 150 to 170 amino acids, 150 to 180 amino acids, 155 to170 amino acids, 160 to 170 amino acids, 155 to 165 amino acids, or 160to 165 amino acids in length. In other embodiments, the therapeuticprotein is 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172 or 173 amino acids in length.

In some embodiments, the protein is a polypeptide which is about 30 to80, 40 to 70, 45 to 55, 35 to 60, 40 to 60, or 35 to 55 amino acids inlength. In other embodiments, the polypeptide is about 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59 or 60 amino acids in length.

In some embodiments, the disclosure provides an antibody or fragmentthereof which specifically binds the therapeutic protein comprising SEQID NO:34 or a variant thereof. In other embodiments, the antibody orfragment thereof does not bind a protein comprising an amino acidsequence identical to SEQ ID NO:3. In still other embodiments, theantibody or fragment thereof binds a protein comprising an amino acidsequence identical to SEQ ID NO:34 but does not bind a proteincomprising an amino acid sequence identical to SEQ ID NO:3.

In some embodiments, the protein increases the barrier function of anepithelial cell layer in an in vitro assay, wherein the increase isrelative to the barrier function in the assay in the absence of theprotein. In other embodiments, the in vitro assay is a transepithelialelectrical resistance (TEER) assay. In still other embodiments, theincrease in barrier function is an increase in electrical resistance ofat least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater thanthe electrical resistance in the assay in the absence of the protein.

In some embodiments, the protein reduces intestinal tissue pathology ina subject administered the protein. In some embodiments, the subject wasinduced to have intestinal tissue damage by treatment with a chemical.In other embodiments, the subject was treated with the chemical dextransodium sulfate (DSS) to induce intestinal tissue damage. In still otherembodiments, the subject is a mammal. In yet other embodiments, theanimal is a rodent. In other embodiments, the subject is a non-humanprimate.

In some embodiments, the therapeutic protein reduces gastrointestinalinflammation in a subject administered the protein. In otherembodiments, the protein reduces intestinal mucosa inflammation in thesubject. In still other embodiments, the protein improves intestinalepithelial cell barrier function or integrity in the subject.

In some embodiments, the protein increases the amount of mucin inintestinal tissue in a subject administered the protein.

In some embodiments, the protein increases intestinal epithelial cellwound healing in a subject administered the protein. In otherembodiments, the protein increases intestinal epithelial cellproliferation in a subject administered the protein.

In some embodiments, the protein prevents or reduces colon shortening ina subject administered the protein.

In some embodiments, the therapeutic protein modulates (i.e., increasesor decreases) a cytokine in the blood, plasma, serum, tissue and/ormucosa of a subject administered the protein.

In some embodiments, the disclosure provides polynucleotides encoding anovel protein therapeutic and methods of expressing said nucleic acidsin a host cell. In a particular embodiment, the polynucleotide comprisesa sequence which encodes a protein that is at least about 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or 100%identical to SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQID NO:40, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQID NO:49. In other embodiments, the polynucleotide comprises a sequencewhich encodes a protein that is at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, or 99.7% identical to SEQ ID NO:42 or SEQ ID NO:44and less than 100% identical to SEQ ID NO:34. In still otherembodiments, the polynucleotide encodes a protein which is anon-naturally occurring variant of SEQ ID NO:1 or SEQ ID NO:3. In stillother embodiments, the polynucleotide is codon-optimized for expressionin a recombinant host cell. In yet other embodiments, the polynucleotideis codon-optimized for expression in E. coli.

In some embodiments, the disclosure provides a nucleic acid whichcomprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:41 or SEQ ID NO:43. In other embodiments,the nucleic acid comprises a sequence which is at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO:43 and less than 100% identical to SEQ ID NO:35.In still other embodiments, the nucleic acid comprises a sequence whichis a non-naturally occurring variant of SEQ ID NO:4.

In some embodiments, the protein is a recombinant protein which issubstantially purified and which is chemically modified at theN-terminus and/or the C-terminus. In other embodiments, the N-terminusof the protein is chemically modified by acetylation. In still otherembodiments, the C-terminus is chemically modified by amidation.

In some embodiments, the protein is a recombinant protein which issubstantially purified and which is pegylated.

In some embodiments, the protein is a recombinant protein which issubstantially purified and which is modified by glycosylation,ubiquitination, nitrosylation, methylation, acetylation, or lipidation.

In some embodiments, the protein is fused to a second protein. In otherembodiments, the second protein is an immunoglobulin Fc domain or ahuman serum albumin protein domain.

In some aspects, the disclosure provides a pharmaceutical compositioncomprising: a therapeutic protein comprising an amino acid sequencehaving at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 100% sequence identity toSEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49and a pharmaceutically acceptable carrier. In some embodiments, thetherapeutic protein is purified or substantially purified. In someembodiments, the protein comprises the amino acid sequence of SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49. In analternative embodiment, the protein does not comprise a sequence whichis identical to SEQ ID NO:34 or SEQ ID NO:36 or the protein is anon-naturally occurring variant of SEQ ID NO:3. In yet otherembodiments, the protein comprises an amino acid sequence of SEQ IDNO:36 or SEQ ID NO:44. In some embodiments, the pharmaceuticalcomposition is useful for treating an inflammatory bowel disease.

In some embodiments, the pharmaceutical composition is formulated forrectal, parenteral, intravenous, topical, oral, dermal, transdermal, orsubcutaneous administration. In other embodiments, the pharmaceuticalcomposition is a liquid, a gel, or a cream. In still other embodiments,the pharmaceutical composition is a solid composition comprising anenteric coating.

In some embodiments, the pharmaceutical composition is formulated toprovide delayed release. In other embodiments, the delayed release isrelease into the gastrointestinal tract. In yet other embodiments, thedelayed release is into the mouth, the small intestine, the largeintestine, and/or the rectum.

In some embodiments, the pharmaceutical composition is formulated toprovide sustained release. In other embodiments, the sustained releaseis release into the gastrointestinal tract. In yet other embodiments,the sustained release is into the mouth, the small intestine, the largeintestine, and/or the rectum. In still other embodiments, the sustainedrelease composition releases the therapeutic formulation over a timeperiod of about 1 to about 20 hours, about 1 to about 10 hours, about 1to about 8 hours, about 4 to about 12 hours, or about 5 to about 15hours.

In some embodiments, the pharmaceutical composition further comprises asecond therapeutic agent. In other embodiments, the second therapeuticagent is selected from the group consisting of an anti-diarrheal, a5-aminosalicylic acid compound, an anti-inflammatory agent, anantibiotic, an anti-cytokine agent, an anti-inflammatory cytokine agent,a steroid, a corticosteroid, an immunosuppressant, a JAK inhibitor, ananti-integrin biologic, an anti-IL12/23R biologic, and a vitamin.

As aforementioned, these novel protein therapeutics are able to promoteepithelial barrier function and integrity in a subject. Additionally,the therapeutic effect of the proteins includes suppression of aninflammatory immune response in an IBD individual. Thus, the disclosureprovides detailed guidance for methods of utilizing the therapeuticproteins provided herein to treat a host of gastrointestinalinflammatory conditions, and disease states involving compromisedgastrointestinal epithelial barrier integrity.

In some embodiments, a method for treating an inflammatory bowel diseaseor disorder in a patient in need thereof is provided, the methodcomprising: administering to the patient a pharmaceutical compositioncomprising: i) a therapeutic protein comprising an amino acid sequencehaving at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9%, or 100% sequence identity to SEQ ID NO:34, SEQID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49; and ii) apharmaceutically acceptable carrier. In other embodiments of the method,the protein comprises an amino acid sequence identical to SEQ ID NO:34,SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49. In still otherembodiments, the protein does not comprise an amino acid sequenceidentical to residues 72 to 232 of SEQ ID NO:3 and/or is a non-naturallyoccurring variant of SEQ ID NO:3.

In some embodiments, therapeutic protein is about 100 to 200 aminoacids, 110 to 190 amino acids, 120 to 180 amino acids, 130 to 170 aminoacids, 140 to 170 amino acids, 150 to 170 amino acids, 150 to 180 aminoacids, 155 to 170 amino acids, 160 to 170 amino acids, 155 to 165 aminoacids, or 160 to 165 amino acids in length. In other embodiments, thetherapeutic protein is 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172 or 173 amino acids inlength.

In some embodiments, the patient has been diagnosed with intestinalinflammation. In other embodiments, the intestinal inflammation is inthe small intestine and/or the large intestine. In still otherembodiments, the intestinal inflammation is in the rectum. In stillother embodiments, the patient has been diagnosed with pouchitis.

In some embodiments, the patient has been diagnosed with intestinalulcers. In other embodiments, the patient has been diagnosed withdraining enterocutaneous and/or rectovaginal fistulas.

In some embodiments, the patient has been diagnosed with Crohn's disease(CD). In other embodiments, the CD is mildly active CD. In still otherembodiments, the CD is moderately to severely active CD. In yet otherembodiments, the patient has been diagnosed with pediatric CD.

In some embodiments, the patient has been diagnosed with short bowelsyndrome or irritable bowel syndrome.

In some embodiments, the patient has been diagnosed with mucositis. Inother embodiments, the mucositis is oral mucositis. In still otherembodiments, the mucositis is chemotherapy-induced mucositis, radiationtherapy-induced mucositis, chemotherapy-induced oral mucositis, orradiation therapy-induced oral mucositis. In yet other embodiments, themucositis is gastrointestinal mucositis. In still other embodiments, thegastrointestinal mucositis is mucositis of the small intestine, thelarge intestine, or the rectum.

In some embodiments, the administering to a patient diagnosed with CDresulted in a reduced number of draining enterocutaneous and/orrectovaginal fistulas. In other embodiments, the administering maintainsfistula closure in adult patients with fistulizing disease.

In other embodiments, the patient has been diagnosed with ulcerativecolitis (UC). In other embodiments, the UC is mildly active UC. In stillother embodiments, the UC is moderately to severely active UC. In stillother embodiments, the patient has been diagnosed with pediatric UC.

In some embodiments, the patient is in clinical remission from an IBD.In other embodiments, the patient is in clinical remission from UC,pediatric UC, CD or pediatric CD.

In some embodiments, the patient has an inflammatory bowel disease ordisorder other than Crohn's disease or ulcerative colitis. In otherembodiments, the patient has at least one symptom associated withinflammatory bowel disease.

In some embodiments, the administering reduces gastrointestinalinflammation and/or reduces intestinal mucosa inflammation associatedwith inflammatory bowel disease in the patient. In other embodiments,the administering improves intestinal epithelial cell barrier functionor integrity in the patient.

In some embodiments, after the administering the patient experiences areduction in at least one symptom associated with an inflammatory boweldisease or disorder. In other embodiments, the at least one symptom isselected from the group consisting of abdominal pain, blood in stool,pus in stool, fever, weight loss, frequent diarrhea, fatigue, reducedappetite, tenesmus, and rectal bleeding. In still other embodiments,after the administering the patient experiences reduced frequency ofdiarrhea, reduced blood in stool and/or reduced rectal bleeding.

In some embodiments, the patient has experienced inadequate response toconventional therapy. In other embodiments, the conventional therapy istreatment with an aminosalicylate, a corticosteroid, a thiopurine,methotrexate, a JAK inhibitor, a sphingosine 1-phosphate (SIP) receptorinhibitor, an anti-integrin biologic, an anti-IL12/23R or anti-IL23/p10biologic, and/or an anti-tumor necrosis factor agent or biologic.

In some embodiments, the administering modulates (i.e., increases ordecreases) levels of a cytokine in the blood, plasma, serum, tissueand/or mucosa of the patient.

In some embodiments, the administering increases intestinal epithelialcell wound healing in the patient. In other embodiments, theadministering increases intestinal epithelial cell proliferation in thepatient.

In some embodiments, the administering prevents or reduces colonshortening in the patient.

In some embodiments, the administering comprises rectal, intravenous,parenteral, oral, topical, dermal, transdermal or subcutaneousadministering of the pharmaceutical composition to the patient. In otherembodiments, the administering is to the gastrointestinal lumen.

In some embodiments, the patient is also administered at least onesecond therapeutic agent. In other embodiments, the at least one secondtherapeutic agent is selected from the group consisting of ananti-diarrheal, an anti-inflammatory agent, an antibody, an antibiotic,or an immunosuppressant. In still other embodiments, the at least onesecond therapeutic agent is an aminosalicylate, a steroid, or acorticosteroid. In other embodiments, the at least one secondtherapeutic agent is selected from the group consisting of adalimumab,pegol, golimumab, infliximab, vedolizumab, ustekinumab, tofacitinib, andcertolizumab or certolizumab pegol.

In some aspects, an expression vector is provided, comprising anexogenous polynucleotide that encodes a protein comprising an amino acidsequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:34, SEQID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49.

In some embodiments, the polynucleotide encodes a protein comprising anamino acid sequence having at least 99% or 100% identity to SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49. Inother embodiments, the polynucleotide encodes a protein comprising theamino acid sequence of SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48 or SEQ ID NO:49. In still other embodiments, the polynucleotideencodes a protein comprising an amino acid sequence that is notidentical to SEQ ID NO:36 or SEQ ID NO:43.

In some embodiments, the protein is about 100 to 200 amino acids, 110 to190 amino acids, 120 to 180 amino acids, 130 to 170 amino acids, 140 to170 amino acids, 150 to 170 amino acids, 150 to 180 amino acids, 155 to170 amino acids, 160 to 170 amino acids, 155 to 165 amino acids, or 160to 165 amino acids in length. In other embodiments, the therapeuticprotein is 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172 or 173 amino acids in length.

In some aspects, an expression system is provided, comprising a hostcell and the expression vector comprising the aforementioned exogenouspolynucleotide.

In some embodiments, the host cell is prokaryotic or eukaryotic. Inother embodiments, the host cell is a mammalian cell, a yeast cell or abacterial cell. In still other embodiments, the bacterial cell isEscherichia coli. In yet other embodiments, the mammalian cell is a CHOcell.

In some aspects, a method of producing the protein is provided.

In some embodiments, a polypeptide comprising SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48 or SEQ ID NO:49 is one which was chemicallysynthesized.

In some embodiments, the method for producing the protein comprisestransforming or transfecting the aforementioned host cell with theaforementioned expression vector, culturing the transformed ortransfected host cell under conditions sufficient for the expression ofthe aforementioned protein encoded by the aforementioned exogenouspolynucleotide. In other embodiments, the method further comprisespurifying the protein from the transformed or transfected host cell andculture media.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show restoration, by SG-11, of epithelial barrierintegrity following inflammation induced disruption, as described inExample 2.

FIG. 2 shows effects of SG-11 administration on epithelial cell woundhealing, as described in Example 3.

FIG. 3 shows effects of SG-11 administration on epithelial centricbarrier function readouts in a DSS model of inflammatory bowel disease,as described in Example 4.

FIG. 4 shows effects of SG-11 administration on inflammatory readoutsresponsive to impaired barrier function in a DSS model of inflammatorybowel disease, as described in Example 4.

FIG. 5 shows effects of SG-11 administration on body weight in a DSSmodel of inflammatory bowel disease, as described in Example 4.

FIG. 6 shows effects of SG-11 administration on gross pathology in a DSSmodel of inflammatory bowel disease, as described in Example 4.

FIGS. 7A, 7B and 7C show results from histopathology analysis ofproximal (FIG. 7A), distal (FIG. 7B) and both proximal and distal (FIG.7C) tissue from a DSS model of inflammatory bowel disease, as describedin Example 4.

FIGS. 8A and 8B show effects of SG-11 administration on colon length(FIG. 8A) and colon weight-to-length (FIG. 8B) in a DSS model ofinflammatory bowel disease, as described in Example 4.

FIG. 9 shows epithelial barrier integrity following SG-11 treatment of aDSS model of inflammatory bowel disease, as described in Example 5.

FIG. 10 shows inflammation centric readouts of barrier function in a DSSmodel of inflammatory bowel disease, as described in Example 5.

FIG. 11 shows prevention of weight loss in a DSS model of inflammatorybowel disease, as described in Example 5.

FIG. 12A shows effects of SG-11 administration on colon length in a DSSmodel of inflammatory bowel disease, as described in Example 5.

FIG. 12B shows effects of SG-11 administration on colon weight-to-lengthratio in a DSS model of inflammatory bowel disease, as described inExample 5.

FIGS. 13A, 13B and 13C show results from histopathology analysis ofproximal (FIG. 13A), distal (FIG. 13B) and both proximal and distal(FIG. 13C) tissue from a DSS model of inflammatory bowel disease, asdescribed in Example 5.

FIG. 14 shows results of the multiple sequence alignment analysis ofSG-11 (SEQ ID NO:7) with similar protein sequences from Roseburiaspecies.

FIG. 15 shows effects of conditions from FIG. 15A, FIG. 15B, FIG. 15C,FIG. 15D, FIG. 15E, FIG. 15F, FIG. 15G, FIG. 15H, and FIG. 15I on SG-11stability. See Example 11 for the conditions associated with FIG. 15A toFIG. 15I.

FIG. 16 shows effects of conditions from FIG. 16A, FIG. 16B, FIG. 16C,FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G, FIG. 16H, and FIG. 16I onSG-11V5 stability. See Example 11 for the conditions associated withFIG. 16A to FIG. 16I.

FIG. 17 shows restoration, by SG-11 and an SG-11 variant, of epithelialbarrier integrity following inflammation induced disruption upon, asdescribed in Example 12.

FIG. 18A and FIG. 18B show epithelial barrier integrity followingtreatment of a DSS model of inflammatory bowel disease with SG-11 and avariant of SG-11, as described in Example 13.

FIG. 19A and FIG. 19B show inflammation centric readouts of barrierfunction in a DSS model of inflammatory bowel disease, as described inExample 13.

FIG. 20A and FIG. 20B show effects of treatment with SG-11 or a variantof SG-11 on weight loss in a DSS model of inflammatory bowel disease, asdescribed in Example 13.

FIG. 21 shows effects of administering SG-11 or a variant of SG-11 ongross pathology in a DSS model of inflammatory bowel disease, asdescribed in Example 13.

FIG. 22A and FIG. 22B show effects of treatment with SG-11 or a variantof SG-11 on colon length in a DSS model of inflammatory bowel disease,as described in Example 13.

FIG. 23A and FIG. 23B show effects of treatment with SG-11 or a variantof SG-11 on colon weight-to-length ratio in a DSS model of inflammatorybowel disease, as described in Example 13.

FIG. 24 shows SDS-PAGE and Coomassie blue analysis of a protein productgenerated upon incubation of SG-11 protein in a fecal slurry asdescribed in Example 14.

FIG. 25 shows SDS-PAGE and Coomassie blue analysis of a protein productgenerated upon incubation of SG-11 protein with trypsin as described inExample 14.

FIG. 26 shows SDS-PAGE and Coomassie blue analysis of a protein productgenerated upon incubation of SG-11 protein with trypsin in the presenceor absence of a trypsin inhibitor as described in Example 14.

FIG. 27 shows restoration, by a product of SG-11 protein incubated infecal slurry, of epithelial barrier integrity following inflammationinduced disruption upon, as described in Example 15.

FIG. 28 shows the sequences SEQ ID NOS:1-50.

DETAILED DESCRIPTION

The present disclosure provides novel protein therapeutics that areuseful in the treatment of subjects suffering from symptoms associatedwith gastrointestinal disorders. For example, these proteins can promoteor enhance epithelial barrier function and/or integrity. The protein mayalso suppress the inflammatory immune response in an IBD individual. Theprotein therapeutic provided herein is useful in treating the numerousdiseases that are associated with decreased gastrointestinal epithelialcell barrier function or integrity and inflammation of the intestine.

In the present disclosure, also provided are protein variants that havetherapeutic activity comparable to or superior to the original protein,but wherein the protein variants have enhanced stability through themanufacturing and processing of the protein therapeutic products as wellas under long-term storage conditions.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, molecular biology, celland cancer biology, immunology, microbiology, pharmacology, and proteinand nucleic acid chemistry, described herein, are those well-known andcommonly used in the art. Thus, while the following terms are believedto be well understood by one of ordinary skill in the art, the followingdefinitions are set forth to facilitate explanation of the presentlydisclosed subject matter.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated component, or group of components, but not the exclusion of anyother components, or group of components.

The term “a” or “an” refers to one or more of that entity, i.e., canrefer to a plural referents. As such, the terms “a” or “an,” “one ormore,” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

The term “about” as used herein with respect to % sequence identity, or% sequence homology, of a nucleic acid sequence, or amino acid sequence,means up to and including ±1.0% in 0.1% increments. For example, “about90%” sequence identity includes 89.0%, 89.1%, 89.2%, 89.3%, 89.4%,89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%, 90.1%, 90.2%, 90.3%, 90.4%,90.5%, 90.6%, 90.7%, 90.8%, 90.9%, and 91%. If not used in the contextof % sequence identity, then “about” means±1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, or 10%, depending upon context of the value in question.

As used herein, a “synthetic protein” means a protein that comprises anamino acid sequence that contains one or more amino acids substitutedwith different amino acids relative to a naturally occurring amino acidsequence. That is, a “synthetic protein” comprises an amino acidsequence that has been altered to contain at least one non-naturallyoccurring substitution modification at a given amino acid position(s)relative to a naturally occurring amino acid sequence.

The terms “gastrointestinal” or “gastrointestinal tract,” “alimentarycanal,” and “intestine,” as used herein, may be used interchangeably torefer to the series of hollow organs extending from the mouth to theanus and including the mouth, esophagus, stomach, small intestine, largeintestine, rectum and anus. The terms “gastrointestinal” or“gastrointestinal tract,” “alimentary canal,” and “intestine” are notalways intended to be limited to a particular portion of the alimentarycanal.

The term “SG-21” as used herein refers to a protein comprising the aminoacid sequence of SEQ ID NO:34 and also to variants thereof having thesame or similar functional activity as described herein. Accordingly,SG-21 can refer herein to proteins comprising or consisting of SEQ IDNO:34 or SEQ ID NO:36, or variants thereof. Examples of SG-21 variantsinclude but are not limited to SEQ ID NO:38 (SG-21V1), SEQ ID NO:39(SG-21V2), and SEQ ID NO:40 (SG-21V5). In U.S. provisional patentapplications (62/482,963, filed Apr. 7, 2017; 62/607,706, filed Dec. 19,2017; 62/611,334, filed Dec. 28, 2017, which describe the relatedprotein, SG-11, and each of which is incorporated herein by reference inits entirety, the term “Experimental Protein 1” and variants thereof wasused and is synonymous with SG-11 as used herein or variants thereof.

A “signal sequence” (also termed “presequence,” “signal peptide,”“leader sequence,” or “leader peptide”) refers to a sequence of aminoacids located at the N-terminus of a nascent protein, and which canfacilitate the secretion of the protein from the cell. The resultantmature form of the extracellular protein lacks the signal sequence,which is cleaved off during the secretion process.

The recitations “sequence identity,” “percent identity,” “percenthomology,” or for example, comprising a “sequence 50% identical to,” asused herein, refer to the extent that sequences are identical on anucleotide-by-nucleotide or amino acid-by-amino acid basis, over awindow of comparison. Thus, a “percentage of sequence identity” may becalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, I) or the identical aminoacid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr,Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity.

The phrases “substantially similar” and “substantially identical” in thecontext of at least two nucleic acids or polypeptides typically meansthat a polynucleotide or polypeptide comprises a sequence that has atleast about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, incomparison with a reference polynucleotide or polypeptide. In someembodiments, substantially identical polypeptides differ only by one ormore conservative amino acid substitutions. In some embodiments,substantially identical polypeptides are immunologically cross-reactive.In some embodiments, substantially identical nucleic acid moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

Related (and derivative) proteins encompass “variant” proteins. Variantproteins differ from another (i.e., parental) protein and/or from oneanother by a small number of amino acid residues. A variant may includeone or more amino acid mutations (e.g., amino acid deletion, insertionor substitution) as compared to the parental protein from which it isderived.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, a conservatively modified variant refers to nucleic acidsencoding identical amino acid sequences, or amino acid sequences thathave one or more “conservative substitutions.” An example of aconservative substitution is the exchange of an amino acid in one of thefollowing groups for another amino acid of the same group (see U.S. Pat.No. 5,767,063; Kyte and Doolittle (1982) J Mol. Biol. 157:105-132). (1)Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, Met; (2) Neutralhydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basic: Asn, Gln,His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro;(6) Aromatic: Trp, Tyr, Phe; and (7) Small amino acids: Gly, Ala, Ser.Thus, the term “conservative substitution” with respect to an amino aciddenotes that one or more amino acids are replaced by another, chemicallysimilar residue, wherein said substitution does not generally affect thefunctional properties of the protein. In some embodiments, thedisclosure provides for proteins that have at least one non-naturallyoccurring, conservative amino acid substitution relative to the aminoacid sequence identified in SEQ ID NO:3 or SEQ ID NO:34. Some commonexemplary examples of conservative amino acid substitutions are foundbelow.

The term “amino acid” or “any amino acid” refers to any and all aminoacids, including naturally occurring amino acids (e.g., α-amino acids),unnatural amino acids, modified amino acids, and unnatural ornon-natural amino acids. It includes both D- and L-amino acids. Naturalamino acids include those found in nature, such as, e.g., the 23 aminoacids that combine into peptide chains to form the building-blocks of avast array of proteins. These are primarily L stereoisomers, although afew D-amino acids occur, e.g., in bacterial envelopes and someantibiotics. The 20 “standard,” natural amino acids are listed in theabove tables. The “non-standard,” natural amino acids are pyrrolysine(found in methanogenic organisms and other eukaryotes), selenocysteine(present in many noneukaryotes as well as most eukaryotes), andN-formylmethionine (encoded by the start codon AUG in bacteria,mitochondria and chloroplasts). “Unnatural” or “non-natural” amino acidsare non-proteinogenic amino acids (i.e., those not naturally encoded orfound in the genetic code) that either occur naturally or are chemicallysynthesized. Over 140 unnatural amino acids are known and thousands ofmore combinations are possible. Examples of “unnatural” amino acidsinclude β-amino acids (β3 and β2), homo-amino acids, proline and pyruvicacid derivatives, 3-substituted alanine derivatives, glycinederivatives, ring-substituted phenylalanine and tyrosine derivatives,linear core amino acids, diamino acids, D-amino acids, alpha-methylamino acids and N-methyl amino acids. Unnatural or non-natural aminoacids also include modified amino acids. “Modified” amino acids includeamino acids (e.g., natural amino acids) that have been chemicallymodified to include a group, groups, or chemical moiety not naturallypresent on the amino acid.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature, or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence. As used herein, a “synthetic amino acid sequence” or“synthetic peptide sequence” or “synthetic polypeptide sequence” or“synthetic protein sequence” is an amino acid sequence that is not knownto occur in nature, or that is not naturally occurring. Generally, sucha synthetic amino acid sequence will comprise at least one amino aciddifference when compared to any other naturally occurring amino acidsequence.

For the most part, the names of natural and non-natural aminoacylresidues used herein follow the naming conventions suggested by theIUPAC Commission on the Nomenclature of Organic Chemistry and theIUPAC-IUB Commission on Biochemical Nomenclature as set out in“Nomenclature of α-Amino Acids (Recommendations, 1974)” Biochemistry,14(2), (1975). To the extent that the names and abbreviations of aminoacids and aminoacyl residues employed in this specification and appendedclaims differ from those suggestions, they will be made clear to thereader.

Among sequences disclosed herein are sequences incorporating a “Hy-”moiety at the amino terminus (N-terminus) of the sequence, and either an“—OH” moiety or an “—NH₂” moiety at the carboxy terminus (C-terminus) ofthe sequence. In such cases, and unless otherwise indicated, a “Hy-”moiety at the N-terminus of the sequence in question indicates ahydrogen atom, corresponding to the presence of a free primary orsecondary amino group at the N-terminus, while an “—OH” or an “—NH₂”moiety at the C-terminus of the sequence indicates a hydroxy group or anamino group, corresponding to the presence of an amido (CONH₂) group atthe C-terminus, respectively. In each sequence of the disclosure, aC-terminal “—OH” moiety may be substituted for a C-terminal “—NH₂”moiety, and vice-versa.

The term “NH₂,” as used herein, can refer to a free amino group presentat the amino terminus of a polypeptide. The term “OH,” as used herein,can refer to a free carboxy group present at the carboxy terminus of apeptide. Further, the term “Ac,” as used herein, refers to acetylprotection through acylation of the C- or N-terminus of a polypeptide.In certain peptides shown herein, the NH₂ locates at the C-terminus ofthe peptide indicates an amino group. The term “carboxy,” as usedherein, refers to —CO₂H. The term “cyclized,” as used herein, refers toa reaction in which one part of a polypeptide molecule becomes linked toanother part of the polypeptide molecule to form a closed ring, such asby forming a disulfide bridge or other similar bond.

The term “pharmaceutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the peptides, proteins, or compounds ofthe present disclosure, which are water or oil-soluble or dispersible,which are suitable for treatment of diseases without undue toxicity,irritation, and allergic response; which are commensurate with areasonable benefit/risk ratio, and which are effective for theirintended use. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting an amino groupwith a suitable acid. Representative acid addition salts includeacetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,formate, fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isethionate), lactate, maleate,mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate, and undecanoate. Also, amino groups in thecompounds of the present disclosure can be quaternized with methyl,ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl,diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, andsteryl chlorides, bromides, and iodides; and benzyl and phenethylbromides. Examples of acids which can be employed to formtherapeutically acceptable addition salts include inorganic acids suchas hydrochloric, hydrobromic, sulfuric, and phosphoric, and organicacids such as oxalic, maleic, succinic, and citric. A pharmaceuticallyacceptable salt may suitably be a salt chosen, e.g., among acid additionsalts and basic salts. Examples of acid addition salts include chloridesalts, citrate salts and acetate salts. Examples of basic salts includesalts where the cation is selected among alkali metal cations, such assodium or potassium ions, alkaline earth metal cations, such as calciumor magnesium ions, as well as substituted ammonium ions. Other examplesof pharmaceutically acceptable salts are described in “Remington'sPharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), MarkPublishing Company, Easton, Pa., USA, 1985 (and more recent editionsthereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rdedition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA,2007, and in J. Pharm. Sci. 66: 2 (1977). Also, for a review on suitablesalts, see Handbook of Pharmaceutical Salts: Properties, Selection, andUse by Stahl and Wermuth (Wiley-VCH, 2002). Other suitable base saltsare formed from bases which form non-toxic salts. Representativeexamples include the aluminum, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids andbases may also be formed, e.g., hemisulphate and hemicalcium salts.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule.

As used herein, the term “host cell” refers to a cell or cell line intowhich a recombinant expression vector for production of a polypeptidemay be introduced for expression of the polypeptide.

The terms “isolated,” “purified,” “separated,” and “recovered” as usedherein refer to a material (e.g., a protein, nucleic acid, or cell) thatis removed from at least one component with which it is naturallyassociated, for example, at a concentration of at least 90% by weight,or at least 95% by weight, or at least 98% by weight of the sample inwhich it is contained. For example, these terms may refer to a materialwhich is substantially or essentially free from components whichnormally accompany it as found in its native state, such as, forexample, an intact biological system.

The terms “patient,” “subject,” and “individual” may be usedinterchangeably and refer to either a human or a non-human animal. Theseterms include mammals such as humans, non-human primates, livestockanimals (e.g., bovines, porcines), companion animals (e.g., canines,felines) and rodents (e.g., mice and rats). In certain embodiments, theterms refer to a human patient. In exemplary embodiments, the termsrefer to a human patient that suffers from a gastrointestinalinflammatory condition.

As used herein, “improved” should be taken broadly to encompassimprovement in an identified characteristic of a disease state, saidcharacteristic being regarded by one of skill in the art to generallycorrelate, or be indicative of, the disease in question, as compared toa control, or as compared to a known average quantity associated withthe characteristic in question. For example, “improved” epithelialbarrier function associated with application of a protein of thedisclosure can be demonstrated by comparing the epithelial barrierintegrity of a human treated with a protein of the disclosure, ascompared to the epithelial barrier integrity of a human not treated.Alternatively, one could compare the epithelial barrier integrity of ahuman treated with a protein of the disclosure to the average epithelialbarrier integrity of a human, as represented in scientific or medicalpublications known to those of skill in the art. In the presentdisclosure, “improved” does not necessarily demand that the data bestatistically significant (i.e., p<0.05); rather, any quantifiabledifference demonstrating that one value (e.g., the average treatmentvalue) is different from another (e.g., the average control value) canrise to the level of “improved.”

As used herein, the term “IBD” or “inflammatory bowel disease” refers toconditions in which individuals have chronic or recurring immuneresponse and inflammation of the gastrointestinal (GI) tract. The twomost common inflammatory bowel diseases are ulcerative colitis (UC) andCrohn's disease (CD).

As used herein, the term “therapeutically effective amount” refers to anamount of a therapeutic agent (e.g., a peptide, polypeptide, or proteinof the disclosure), which confers a therapeutic effect on the treatedsubject, at a reasonable benefit/risk ratio applicable to any medicaltreatment. Such a therapeutic effect may be objective (i.e., measurableby some test or marker) or subjective (i.e., subject gives an indicationof, or feels an effect). In some embodiments, “therapeutically effectiveamount” refers to an amount of a therapeutic agent or compositioneffective to treat, ameliorate, or prevent (e.g., delay onset of) arelevant disease or condition, and/or to exhibit a detectabletherapeutic or preventative effect, such as by ameliorating symptomsassociated with the disease, preventing or delaying onset of thedisease, and/or also lessening severity or frequency of symptoms of thedisease. A therapeutically effective amount is commonly administered ina dosing regimen that may comprise multiple unit doses. For anyparticular therapeutic agent, a therapeutically effective amount (and/oran appropriate unit dose within an effective dosing regimen) may vary,for example, depending on route of administration, or in combinationwith other therapeutic agents. Alternatively or additionally, a specifictherapeutically effective amount (and/or unit dose) for any particularpatient may depend upon a variety of factors including the particularform of disease being treated; the severity of the condition orpre-condition; the activity of the specific therapeutic agent employed;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration, route ofadministration, and/or rate of excretion or metabolism of the specifictherapeutic agent employed; the duration of the treatment; and likefactors as is well-known in the medical arts. The current disclosureutilizes therapeutically effective amounts of novel proteins, andcompositions comprising the same, to treat a variety of diseases, suchas: gastrointestinal inflammatory diseases or diseases involvinggastrointestinal epithelial barrier malfunction. The therapeuticallyeffective amounts of the administered protein, or compositionscomprising same, will in some embodiments reduce inflammation associatedwith IBD or repair gastrointestinal epithelial barrier integrity and/orfunction.

As used herein, the term “treatment” (also “treat” or “treating”) refersto any administration of a therapeutic agent (e.g., a peptide,polypeptide, or protein of the disclosure), according to a therapeuticregimen that achieves a desired effect in that it partially orcompletely alleviates, ameliorates, relieves, inhibits, delays onset of,reduces severity of and/or reduces incidence of one or more symptoms orfeatures of a particular disease, disorder, and/or condition (e.g.,chronic or recurring immune response and inflammation of thegastrointestinal (GI) tract); in some embodiments, administration of thetherapeutic agent according to the therapeutic regimen is correlatedwith achievement of the desired effect. Such treatment may be of asubject who does not exhibit signs of the relevant disease, disorderand/or condition and/or of a subject who exhibits only early signs ofthe disease, disorder, and/or condition. Alternatively or additionally,such treatment may be of a subject who exhibits one or more establishedsigns of the relevant disease, disorder and/or condition. In someembodiments, treatment may be of a subject who has been diagnosed assuffering from the relevant disease, disorder, and/or condition. In someembodiments, treatment may be of a subject known to have one or moresusceptibility factors that are statistically correlated with increasedrisk of development of the relevant disease, disorder, and/or condition.

“Pharmaceutical” implies that a composition, reagent, method, and thelike, are capable of a pharmaceutical effect, and also that thecomposition is capable of being administered to a subject safely.“Pharmaceutical effect,” without limitation, can imply that thecomposition, reagent, or method, is capable of stimulating a desiredbiochemical, genetic, cellular, physiological, or clinical effect, in atleast one individual, such as a mammalian subject, for example, a human,in at least 5% of a population of subjects, in at least 10%, in at least20%, in at least 30%, in at least 50% of subjects, and the like.“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopoeia orother generally recognized pharmacopoeia for safe use in animals, andmore particularly safe use in humans. “Pharmaceutically acceptablevehicle” or “pharmaceutically acceptable excipient” refers to a diluent,adjuvant, excipient or carrier with which a protein as described hereinis administered.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a subject that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease, or causing the symptom to develop with lessseverity than in absence of the treatment). “Prevention” or“prophylaxis” may refer to delaying the onset of the disease ordisorder.

The therapeutic pharmaceutical compositions provided herein may compriseone or more natural products. However, in certain embodiments, thetherapeutic pharmaceutical compositions themselves do not occur innature. Further, in certain embodiments, the therapeutic pharmaceuticalcompositions possess markedly different characteristics, as compared toany individual naturally occurring counterpart, or compositioncomponent, which may exist in nature. That is, in certain embodiments,the pharmaceutical compositions provided herein—which comprise atherapeutically effective amount of a purified protein—possess at leastone structural and/or functional property that impart markedly differentcharacteristics to the composition as a whole, as compared to any singleindividual component of the composition as it may exist naturally. Thecourts have determined that compositions comprising natural products,which possess markedly different characteristics as compared to anyindividual component as it may exist naturally, are statutory subjectmatter. Thus, the provided therapeutic pharmaceutical compositions as awhole possess markedly different characteristics. These characteristicsare illustrated in the data and examples provided herein.

Details of the disclosure are set forth herein. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, illustrative methodsand materials are now described. Other features, objects, and advantagesof the disclosure will be apparent from the description and from theclaims.

Therapeutic Proteins from the Microbiome—Overview of the Disclosure

Numerous diseases and disorders are associated with decreasedgastrointestinal epithelial cell barrier function or integrity. Thesediseases and disorders are multifaceted and present diagnostically in amyriad of ways. One such disease is inflammatory bowel disease (IBD),the incidence and prevalence of which is increasing with time and indifferent regions around the world, indicating its emergence as a globaldisease. (Molodecky et al., Gastroenterol 142:46-54, 2012). IBD is acollective term that describes conditions with chronic or recurringimmune response and inflammation of the gastrointestinal (GI) tract. Thetwo most common inflammatory bowel diseases are ulcerative colitis (UC)and Crohn's disease (CD). Both are marked by an abnormal response of theGI immune system. Normally, immune cells protect the body frominfection. In people with IBD, however, this immune system mistakesfood, bacteria, and other materials in the intestine for pathogens andan inflammatory response is launched into the lining of the intestines,creating chronic inflammation. When this happens, the patientexperiences the symptoms of IBD.

IBD involves chronic inflammation of all, or part, of the digestivetract. Both UC and CD usually involve, for example, severe diarrhea,abdominal pain, fatigue, and weight loss. IBD and associated disorderscan be debilitating and sometimes lead to life-threateningcomplications.

With respect to intestinal barrier integrity, loss of integrity of theintestinal epithelium plays a key pathogenic role in IBD. Maloy, KevinJ.; Powrie, Fiona, 2011 Nature. 474 (7351): 298-306: Coskun, 2014, FrontMed (Lausanne), 1:24; Martini et al., 2017, Cell Mol GastroenterolHepatol, 4:33-46. It is hypothesized that detrimental changes in theintestinal microbiota induce an inappropriate or uncontrolled immuneresponse that results in damage to the intestinal epithelium. Breachesin this critical intestinal epithelium barrier allow furtherinfiltration of microbiota that, in turn, elicit further immuneresponses. Thus, IBD is a multifactorial disease that is driven in partby an exaggerated immune response to gut microbiota that can causedefects in epithelial barrier function.

Microbiome profiling of IBD patients has revealed distinct profiles suchas increased Proteobacteria, including adherent-invasive E. coli, oftenat the expense of potentially beneficial microbes such as Roseburia spp(Machiels et al., 2014, Cut, 63:1275-1283; Patterson et al., 2017, FrontImmunol, 8:1166; Shawki and McCole, 2017, Cell Mol GastroenterolHepatol, 3:41-50). Moreover, a decrease in Roseburia hominis was linkedwith dysbiosis in patients with ulcerative colitis. IBD affectedindividuals have been found to have 30-50 percent reduced biodiversityof commensal bacteria, such as decreases in Firmicutes (namelyLachnospiraceae) and Bacteroidetes. Further evidence of the role of gutflora in the cause of inflammatory bowel disease is that IBD affectedindividuals are more likely to have been prescribed antibiotics in the2-5 year period before their diagnosis than unaffected individuals. See,Aroniadis O C, Brandt L J, “Fecal microbiota transplantation: past,present and future,” (2013) Curr. Opin. Gastroenterol. 29 (1) (2013):79-84.

Protective bacterial communities, probiotics and bacterially derivedmetabolites have been demonstrated to improve disease in variousclinical and pre-clinical studies. For example, fecal microbial transfer(FMT) experiments have shown some success in IBD patients, althoughchallenges still exist with FMT (Moayyedi et al., 2015,Gastroenterology, 149:102-109 e106; Qazi et al., 2017, Gut Microbes,8:574-588; Narula et al., 2017, Inflamm Bowel Dis, 23:1702-1709). Inother studies treatment with probiotics including VSL#3, Lactobacillusspp. and Bifidobacterium spp. have also shown to have beneficial effectsin humans and animal models (Srutkova et al., 2015, PLoS One,10:e0134050; Pan et al., 2014, Benef Microbes, 5:315-322; Huynh et al.,2009, Inflamm Bowel Dis, 15:760-768; Bibiloni et al., 2005, Am JGastroenterol, 100:1539-1546). Furthermore, bacterial products such asp40 from L. rhamnosus GG and Amuc-1100 from A. muciniphila have beenshown to promote barrier function and protect in animal models of IBDand metabolic disease, receptively (Yan et al., 2011, J Clin Invest,121:2242-2253; Plovier et al., Nat Med, 23:107-113).

While uses of live microbial populations to treat diseases isincreasingly common, such methods rely on the ability of theadministered bacteria to survive in the host or patient and to interactwith the host tissues in a beneficial and therapeutic way. Analternative approach, provided here, is to identify microbially-encodedproteins and variants thereof which can affect cellular functions in thehost and provide therapeutic benefit. Such proteins can be administered,for example, as pharmaceutical compositions comprising a substantiallyisolated or purified therapeutic, bacterially-derived protein or as alive biotherapeutic (bacterium) engineered to express the therapeuticprotein as an exogenous protein. Moreover, methods of treatmentcomprising administration of the therapeutic protein are not limited tothe gut (small intestine, large intestine, rectum) but may also includetreatment of other disorders within the alimentary canal such as oralmucositis.

To identify microbially-derived proteins which may have therapeuticapplication in gastrointestinal inflammatory disorders, fecal samplesfrom humans who were healthy or who were diagnosed with UC or CD wereanalyzed to determine the microbial compositions of fecal samplescollected from these individuals. A comparison of the bacterial profilesfrom healthy vs. diseased subjects identified bacteria that were eitherlikely to be beneficial (greater numbers in healthy vs. diseased) ordetrimental (lower numbers in healthy vs. diseased). Among the bacterialspecies identified as beneficial was Roseburia hominis, consistent withstudies referenced above. Extensive bioinformatics analysis was thenperformed to predict proteins encoded by the bacterium and then toidentify those proteins which are likely to be secreted by thebacterium. Proteins which were predicted to be secreted proteins werethen characterized using a series of in vitro assays to study the effectof each protein on epithelial barrier integrity, cytokine productionand/or release, and wound healing. Proteins identified as functioning toincrease epithelial barrier integrity were then assessed in an in vivomouse model for colitis. One such protein, identified herein as “SG-11,”demonstrated both in vitro and in vivo activity indicative of itsability to provide therapeutic benefit for increasing epithelial barrierintegrity and for treating diseases and disorders associated withepithelial barrier integrity as well as treating inflammatorygastrointestinal diseases such as IBDs. The amino acid andpolynucleotide sequences of SG-11 and variants thereof, as well asfunctional activity of the SG-11 protein and variants thereof wasdescribed in U.S. provisional patent applications 62/482,963, filed Apr.7, 2017; 62/607,706, filed Dec. 19, 2017; 62/611,334, filed Dec. 28,2017 and International Publication No. WO 2018/187682 filed on Apr. 6,2018. The disclosure of each of these applications is incorporatedherein by reference in their entirety. The SG-11 protein, variantsthereof, and functional activity are summarized below and in Examples1-13.

The SG-11 Protein

The SG-11 protein is encoded within a 768 nucleotide sequence (SEQ IDNO:2) present in the genome of Roseburia hominis. A complete genomicsequence for R. hominis strain can be found at GenBank accession numberCP003040 (the sequence incorporated herein by reference in itsentirety). A 16S rRNA gene sequence for the Roseburia hominis strain canbe found at GenBank accession number AJ270482. The full-length proteinencoded by the R. hominis genomic sequence is 256 amino acids in length(SEQ ID NO:1), wherein residues 1-25 are predicted to be a signalpeptide which is cleaved in vivo to produce a mature protein of 232amino acids (SEQ ID NO:3; encoded by SEQ ID NO:4). Recombinant SG-11 canbe expressed with an N-terminal methionine (encoded by the codon ATG) toproduce a mature protein of 233 amino acids (SEQ ID NO:7).

SG-11 was recombinantly expressed in different commercially availableand routinely used expression vectors. For example, SG-11 (a proteincomprising SEQ ID NO:3), was expressed using a pGEX expression vectorwhich expresses the protein of interest with a GST tag and protease sitewhich is cleaved after expression and purification, a pET-28 expressionvector which adds an N-terminal FLAG tag, and a pD451 expression vectorwhich was used to express the SG-11 protein consisting of SEQ ID NO:7and having no N-terminal tag. Experiments performed and repeated withthese proteins showed that the minor N-terminal and/or C-terminalvariations resulting from the use of the different protein expressionsystems and DNA constructs retained equivalent functional activity in invivo and in vitro assays. It is understood that unless otherwiseindicated, the term “SG-11” refers herein to the amino acid sequencedepicted herein as SEQ ID NO:3 and alternative versions of the proteincomprising the amino acid sequence of SEQ ID NO:3 such as proteinscomprising the amino acid sequence of SEQ ID NO:3 and a startmethionine; and/or a protein comprising the amino acid sequence of SEQID NO:3 and a tag; and/or a protein comprising the amino acid sequenceof SEQ ID NO:3 and a fusion partner (including but not limited to SEQ IDNO:1, SEQ ID NO:5, SEQ ID NO:7). SG-11 variants can include variationsin amino acid residues (substitution, insertion, deletion) as well asmodifications such as fusion constructs and post-translationalmodifications (phosphorylation, glycosylation, etc.). Some exemplaryembodiments of the SG-11 protein and encoding nucleic acids are providedin Table 1 below.

TABLE 1 Amino Acid Sequence Encoding Nucleic Acid Sequence SEQ ID NO: 1SEQ ID NO: 2 MKRLVCTVCSVLLCAGLL ATGAAGAGATTAGTGTGCACGGTCTGCAGTGTACTGTSGCGTSLEGEESVVYVGK TGTGTGCGGGACTTCTCTCCGGATGCGGTACCT KGVIASLDVETLDQSYYDECGCTGGAGGGAGAGGAAAGTGTCGTGTACGTGGGAA TELKSYVDAEVEDYTAEHAGAAAGGCGTGATAGCGTCGCTGGATGTGGAGAC GKNAVKVESLKVEDGVAKGCTCGATCAGTCCTACTACGATGAGACGGAACTGAA LKMKYKTPEDYTAFNGIELGTCCTATGTGGATGCAGAGGTGGAAGATTACACC YQGKVVASLAAGYVYDGGCGGAGCATGGTAAAAATGCAGTCAAGGTGGAGAGC EFARVEEGKVVGAATKQDCTTAAGGTGGAAGACGGTGTGGCGAAGCTTAAGA IYSEDDLKVAIIRANTDVKTGAAGTACAAGACACCGGAGGATTATACCGCATTTA VDGEICYVSCQNVKLTGKATGGAATTGAACTCTATCAGGGGAAAGTCGTTGC DSVSIRDGYYLETGSVTASTTCCCTGGCGGCAGGATACGTCTACGACGGGGAGTT VDVTGQESVGTEQLSGTECGCCCGCGTGGAGGAAGGCAAGGTTGTGGGAGCT QMEMTGEPVNADDTEQTEGCCACAAAACAGGATATTTACTCTGAGGATGATTTG AAAGDGSFETDVYTFIVYKAAAGTTGCCATCATCCGTGCCAATACGGATGTGA AGGTGGACGGTGAGATCTGCTATGTCTCCTGTCAGAATGTGAAGCTGACCGGAAAAGACAGTGTGTCGAT CCGTGACGGATATTATCTTGAGACGGGAAGCGTGACGGCATCCGTGGATGTGACCGGACAGGAGAGCGTC GGGACCGAGCAGCTTTCGGGAACCGAACAGATGGAGATGACCGGGGAGCCGGTGAATGCGGATGATACCG AGCAGACAGAGGCGGCGGCCGGTGACGGTTCGTTCGAGACAGACGTATATACTTTCATTGTCTACAAA SEQ ID NO: 3 SEQ ID NO: 4LEGEESVVYVGKKGVIASL TTGGAGGGTGAAGAGTCTGTTGTCTATGTGGGTAAGDVETLDQSYYDETELKSY AAAGGTGTGATCGCGTCCCTGGACGTCGAGACTCTGVDAEVEDYTAEHGKNAVK GACCAGTCTTACTATGATGAAACCGAGCTGAAGTCGVESLKVEDGVAKLKMKYK TATGTGGACGCCGAAGTTGAGGATTACACGGCCGAGTPEDYTAFNGIELYQGKVV CACGGCAAAAATGCCGTCAAAGTTGAGAGCTTGAAAASLAAGYVYDGEFARVEE GTTGAGGACGGCGTGGCAAAGCTGAAGATGAAATACGKVVGAATKQDIYSEDDL AAGACCCCAGAGGACTACACGGCGTTCAATGGTATCKVAIIRANTDVKVDGEICY GAGCTGTATCAGGGCAAAGTCGTCGCATCCCTGGCAVSCQNVKLTGKDSVSIRDG GCGGGCTATGTGTACGACGGTGAGTTTGCGCGCGTCYYLETGSVTASVDVTGQES GAAGAAGGCAAAGTTGTGGGTGCGGCTACGAAACAAVGTEQLSGTEQMEMTGEP GATATCTACAGCGAAGATGACCTGAAAGTCGCGATTVNADDTEQTEAAAGDGSF ATTCGTGCTAACACCGATGTTAAAGTTGATGGCGAG ETDVYTFIVYKATTTGCTACGTTAGCTGTCAAAACGTAAAGCTGACGGGTAAAGATAGCGTGAGCATTCGTGATGGCTATTATCTGGAAACCGGTAGCGTTACGGCGAGCGTCGATGTTACCGGTCAAGAGAGCGTGGGTACCGAACAGCTGAGCGGCACCGAACAGATGGAAATGACCGGTGAACCGGTTAA CGCAGACGACACGGAACAAACCGAAGCCGCGGCAGGCGACGGTAGCTTCGAGACTGACGTGTACACCTTTAT CGTGTACAAG SEQ ID NO: 7SEQ ID NO: 8 MLEGEESVVYVGKKGVIA ATGTTGGAGGGTGAAGAGTCTGTTGTCTATGTGGGTASLDVETLDQSYYDETELKS AGAAAGGTGTGATCGCGTCCCTGGACGTCGAGACTCYVDAEVEDYTAEHGKNAV TGGACCAGTCTTACTATGATGAAACCGAGCTGAAGTKVESLKVEDGVAKLKMKY CGTATGTGGACGCCGAAGTTGAGGATTACACGGCCGKTPEDYTAFNGIELYQGKV AGCACGGCAAAAATGCCGTCAAAGTTGAGAGCTTGAVASLAAGYVYDGEFARVE AAGTTGAGGACGGCGTGGCAAAGCTGAAGATGAAATEGKVVGAATKQDIYSEDD ACAAGACCCCAGAGGACTACACGGCGTTCAATGGTALKVAIIRANTDVKVDGEIC TCGAGCTGTATCAGGGCAAAGTCGTCGCATCCCTGGCYVSCQNVKLTGKDSVSIRD AGCGGGCTATGTGTACGACGGTGAGTTTGCGCGCGTGYYLETGSVTASVDVTGQ CGAAGAAGGCAAAGTTGTGGGTGCGGCTACGAAACAESVGTEQLSGTEQMEMTG AGATATCTACAGCGAAGATGACCTGAAAGTCGCGATEPVNADDTEQTEAAAGDG TATTCGTGCTAACACCGATGTTAAAGTTGATGGCGAG SFETDVYTFIVYKATTTGCTACGTTAGCTGTCAAAACGTAAAGCTGACGGGTAAAGATAGCGTGAGCATTCGTGATGGCTATTATCTGGAAACCGGTAGCGTTACGGCGAGCGTCGATGTTACCGGTCAAGAGAGCGTGGGTACCGAACAGCTGAGCGGCACCGAACAGATGGAAATGACCGGTGAACCGGTTAA CGCAGACGACACGGAACAAACCGAAGCCGCGGCAGGCGACGGTAGCTTCGAGACTGACGTGTACACCTTTAT CGTGTACAAG SEQ ID NO: 9SEQ ID NO: 10 MDYKDDDDKGSSHMLEG ATGGACTACAAAGACGATGACGACAAGGGCAGCAGCEESVVYVGKKGVIASLDVE CATATGCTGGAGGGAGAGGAAAGTGTCGTGTACGTGTLDQSYYDETELKSYVDA GGAAAGAAAGGCGTGATAGCGTCGCTGGATGTGGAGEVEDYTAEHGKNAVKVES ACGCTCGATCAGTCCTACTACGATGAGACGGAACTGLKVEDGVAKLKMKYKTPE AAGTCCTATGTGGATGCAGAGGTGGAAGATTACACCDYTAFNGIELYQGKVVASL GCGGAGCATGGTAAAAATGCAGTCAAGGTGGAGAGCAAGYVYDGEFARVEEGKV CTTAAGGTGGAAGACGGTGTGGCGAAGCTTAAGATGVGAATKQDIYSEDDLKVAI AAGTACAAGACACCGGAGGATTATACCGCATTTAATIRANTDVKVDGEICYVSCQ GGAATTGAACTCTATCAGGGGAAAGTCGTTGCTTCCCNVKLTGKDSVSIRDGYYLE TGGCGGCAGGATACGTCTACGACGGGGAGTTCGCCCTGSVTASVDVTGQESVGTE GCGTGGAGGAAGGCAAGGTTGTGGGAGCTGCCACAAQLSGTEQMEMTGEPVNAD AACAGGATATTTACTCTGAGGATGATTTGAAAGTTGCDTEQTEAAAGDGSFETDV CATCATCCGTGCCAATACGGATGTGAAGGTGGACGG YTFIVYKTGAGATCTGCTATGTCTCCTGTCAGAATGTGAAGCTGACCGGAAAAGACAGTGTGTCGATCCGTGACGGATATTATCTTGAGACGGGAAGCGTGACGGCATCCGTGGATGTGACCGGACAGGAGAGCGTCGGGACCGAGCAGCTTTCGGGAACCGAACAGATGGAGATGACCGGGGAGCCGGTGAATGCGGATGATACCGAGCAGACAGAGGCGGCGGCCGGTGACGGTTCGTTCGAGACAGACGTATATACTT TCATTGTCTACAAA

Epithelial Barrier Function in Disease

Studies in recent years have identified a major role of both genetic andenvironmental factors in the pathogenesis of IBD. Markus Neurath,“Cytokines in Inflammatory Bowel Disease,” Nature Reviews Immunology,Vol. 14., 329-342 (2014). A combination of these IBD risk factors seemsto initiate detrimental changes in epithelial barrier function, therebyallowing the translocation of luminal antigens (for example, bacterialantigens from the commensal microbiota) into the bowel wall. Id.Subsequently, aberrant and excessive responses, such as increasedpro-inflammatory cytokine release, to such environmental triggers causesubclinical or acute mucosal inflammation in a genetically susceptiblehost. Id. Thus, the importance of proper epithelial barrier function inIBD is apparent, for in subjects that fail to resolve acute intestinalinflammation, chronic intestinal inflammation develops that is inducedby the uncontrolled activation of the mucosal immune system. Inparticular, mucosal immune cells, such as macrophages, T cells, and thesubsets of innate lymphoid cells (ILCs), seem to respond to microbialproducts or antigens from the commensal microbiota by, e.g., producingcytokines that can promote chronic inflammation of the gastrointestinaltract. Consequently, restoring proper epithelial barrier function topatients may be critical in resolving IBD.

The therapeutic activity of SG-11 was identified in part by itsbeneficial effects on epithelial barrier function both in vitro and invivo. SG-11 was shown to be active in increasing epithelial barrierintegrity as shown by an in vitro trans-epithelial electrical resistance(TEER) assay (see Example 2). A TEER assay is a well-known method formeasuring effects on the structural and functional integrity of anepithelial cell layer (Srinivasan et al., 2015, J Lab Autom, 20:107-126;Beduneau et al., 2014, Eur J Pharm Biopharm, 87:290-298; Zolotarevsky etal., 2002, Gastroenterology, 123:163-172, Dewi, et al., 2004, J. Virol.Methods. 121:171-180, Dewi, et al., 2004, J. Virol. Methods. 121:171-180, and Mandic, et al., 2004, Clin. Exp. Metast. 21:699-704). Theassay performed and described herein consists of an epithelial monolayermade up of enterocyte and goblets cells to more accurately model thestructural and functional components of the intestinal epithelium. Thecells are cultured until tight junction formation occurs and barrierfunction capacity is assessed by a measurement of trans-epithelialelectrical resistance. Upon addition of an insult, such as heat killedE. coli, there is a decrease in electrical resistance across theepithelial layer. Control reagents useful in the TEER assay includestaurosporine and a myosin light chain kinase inhibitor. Staurosporineis a broad spectrum kinase inhibitor, originating from Streptomycesstaurosporeus, which induces apoptosis. This reagent disrupts about 98%of the gap junctions leading to a decrease in electrical resistance in aTEER assay. Myosin light chain kinase (MLCK) is the terminal effector ina signaling cascade induced by pro-inflammatory cytokines, which resultsin contraction of the perijunctional actomyosin ring, resulting inseparation of the gap junctions. By inhibiting MLCK, disruption of tightjunctions is prevented. MLCK inhibitor in a TEER assay should reduce orprevent the reduction of electrical resistance in a TEER assay.

As noted above, IBDs and other gastrointestinal disorders includinginflammatory disorders are believed to be associated with decreasedepithelial barrier integrity which leads inter alia to bacteria crossingthe barrier and inciting an immune response. Example 3 shows that SG-11protein can enhance or facilitate epithelial wound healing, an activitythat can play a role in the maintenance or repair of and epithelialbarrier such as an intestinal or mucosal epithelial barrier.

In view of the effect of SG-11 to repair barrier function integrity,SG-11 was analyzed in vivo for its ability to reduce damage in a rodentmodel of IBD. Examples 4 and 5 (SG-11) and 13 (SG-11 variant) describestudies done using a DSS (dextran sodium sulfate) animal model, a modelwell accepted for the study of agents on IBDs (Chassaign et al., 2014,Curr Protoc Imunol, 104:Unit-15.25; Kiesler et al., 2015, Cell MolGastroenterol Hepatol). DSS is a sulfated polysaccharide that isdirectly toxic to colonic epithelium and causes epithelial cell injuryleading to loss of barrier function due to disrupted gap junctions. Inthese experiments, animals were treated with SG-11 either prior to(Example 4) or after (Example 5) induction of colitis in the mouse. As apositive control, the mice were also treated with Gly2-GLP2, a stableanalog of glucagon-like peptide 2 (GLP2). Gly2-GLP2 is known to promoteepithelial cell growth and reduce colonic injury in experiment mousecolitis models. Results of the DSS studies show that SG-11 protein waseffective in reducing weight loss in DSS models, an important indicatorof clinical efficacy for IBD therapeutics. SG-11 treatment also reducedscores in gross pathology and intestinal histopathology analyses.

It is noted that while SG-11 treatment improved the 4Kda-FITC intestinalpermeability readout and reduced serum levels of LPS binding protein(LBP—a marker of LPS exposure) in Example 7, no significant effects upontreatment with SG-11 or Gly2-GLP2 were observed in Example 8. This isnot surprising when considering that animals in Example 8 were treatedwith DSS for 7 days prior to replacement with normal drinking water andtreatment with SG-11 or Gly2-GLP2. This prior exposure to DSS results indamage to the intestinal epithelium, translocation of LPS across adisrupted epithelial barrier, and induction of LBP secretion. However,based on 4KDa-FITC dextran measurements, epithelial barrier repairappears to occur rapidly, within 3-4 days, following replacement of DSSwith normal drinking water (data not shown, FIG. 12). Accordingly, it isdifficult to detect improvements in 4KDa-FITC permeability readouts intreated vs. untreated animals at the time of measurement (after 6 daysof treatment). Additionally, levels of LBP in the serum may beindependent of barrier function repair in animals exposed to DSS for anextended period of time prior to therapeutic treatment (Example 8). Forinstance, hepatocytes activated by translocating LPS during the DSSexposure produce and secrete large amounts of LPB. Accordingly, andwithout being bound by theory, the short time period of the study maynot allow sufficient time for inactivation of the hepatocytes andclearance of LBP from the serum of the DSS-treated animals. It isconsidered, therefore, that continuation of the study with measurementof serum LBP at later time points would show a decrease in serum LBPlevels, however, the decrease in serum LBP may be similar in bothtreated and untreated animals if barrier function is restored in bothanimals before LBP can be cleared from the serum.

Amino Acid Variants of SG-11

In view of the therapeutic value of SG-11 and its use for treatingdisease, the protein was further characterized and its sequence modifiedto change its primary structure in ways that would optimizepharmaceutical formulation and long-term storage of the protein.

As described in Example 6, SG-11 (SEQ ID NO:7) was used to perform aBLAST search using the GenBank non-redundant protein database toidentify proteins with similar amino acid sequences and which may befunctional homologs or have function(s) similar to those of SG-11. Threesuch proteins were identified and the predicted mature sequence of each(without an N-terminal signal peptide) was aligned with SEQ ID NO:3 toidentify regions and individual positions within the proteins which wererelatively conserved. These 3 proteins are disclosed herein as SEQ IDNO:21 (derived from GenBank Acc. No. WP_006857001), SEQ ID NO:22(derived from GenBank Acc. No. WP_075679733), and SEQ ID NO:23 (derivedfrom GenBank Acc. No. WP_055301040), (FIG. 14) and accordingly, providedherein are pharmaceutical compositions comprising 1 of these 3 proteinsor a variant or fragment thereof, as well as methods for treatingdiseases associated with barrier function disorders and/orgastrointestinal diseases or disorders comprising administering to asubject in need thereof a pharmaceutical composition comprising any oneof SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 or a variant or fragmentthereof. In some embodiments, provided is a protein comprising an aminoacid sequence which is at least 90%, 95%, 97%, 98% or 99% identical tothe sequence of residues 73 to 227 of SEQ ID NO:21 or a fragmentthereof, residues 72 to 215 of SEQ ID NO:22 or a fragment thereof, orresidues 72 to 236 of SEQ ID NO:23 or a fragment thereof.

An embodiment of SG-11V5 and an encoding nucleic acid sequence isprovided in Table 2 below.

TABLE 2 Amino Acid Sequence Encoding Nucleic Acid SequenceSEQ ID NO: 19 (SG-11V5) SEQ ID NO: 20 MLEGEESVVYVGKKGVIAATGTTGGAGGGTGAAGAGTCTGTTGTCTATGTGGGTA SLDVETLDQSYYDETELKSAGAAAGGTGTGATCGCGTCCCTGGACGTCGAGACTC YVDAEVEDYTAEHGKSAVTGGACCAGTCTTACTATGATGAAACCGAGCTGAAGT KVESLKVEDGVAKLKMKYCGTATGTGGACGCCGAAGTTGAGGATTACACGGCCG KTPEDYTAFSGIELYQGKVAGCACGGCAAATCCGCCGTCAAAGTTGAGAGCTTGA VASLAAGYVYDGEFARVEAAGTTGAGGACGGCGTGGCAAAGCTGAAGATGAAAT EGKVVGAATKQDIYSEDDACAAGACCCCAGAGGACTACACGGCGTTCAGCGGTA LKVAIIRANTDVKVDGEIVTCGAGCTGTATCAGGGCAAAGTCGTCGCATCCCTGGC YVSSQNVKLTGKDSVSIRDAGCGGGCTATGTGTACGACGGTGAGTTTGCGCGCGT GYYLETGSVTASVDVTGQCGAAGAAGGCAAAGTTGTGGGTGCGGCTACGAAACA ESVGTEQLSGTEQMEMTGAGATATCTACAGCGAAGATGACCTGAAAGTCGCGAT EPVNADDTEQTEAAAGDGTATTCGTGCTAACACCGATGTTAAAGTTGATGGCGAG SFETDVYTFIVYKATTGTGTACGTTAGCAGCCAAAACGTAAAGCTGACGGGTAAAGATAGCGTGAGCATTCGTGATGGCTATTATCTGGAAACCGGTAGCGTTACGGCGAGCGTCGATGTTACCGGTCAAGAGAGCGTGGGTACCGAACAGCTGAGCGGCACCGAACAGATGGAAATGACCGGTGAACCGGTTA ACGCAGACGACACGGAACAAACCGAAGCCGCGGCAGGCGACGGTAGCTTCGAGACTGACGTGTACACCTTTA TCGTGTACAAG

In the interest of enhancing the stability of SG-11 proteins for use inpharmaceutical formulations and clinical applications, studies wereperformed to identify and characterize post translational modificationsof purified SG-11 protein. These experiments are described in Examples7-9. Such analysis shows that the SG-11 protein can undergo at least thepost-translational modifications (PTMs) of methionine oxidation, andasparagine deamidation. Moreover, experiments described in Example 10show the cysteines in SG-11 are unlikely to form disulfide bonds in thenative, functional conformation of the active protein, suggesting thatthe free sulfhydryl groups in SG-11 may cause aggregation in a solutioncontaining the purified protein. Based on these stability studies anddespite the conserved nature of the residues in SG-11 as seen in themultiple sequence alignment (FIG. 14), it was decided to test whether ornot the cysteines at positions 147 and/or 151 (with reference to SEQ IDNO:7) could be substituted with a different amino acid. Also,substitution of conserved asparagines at positions 53 and 83 wereconsidered. In an exemplary embodiment, the SG-11 sequence of SEQ IDNO:7 is modified to introduce the substitutions of C147V and C151S togenerate SEQ ID NO:11 (SG-11V1). The Cl 47V and C151S substitutions arealso present in the provided SG-11 variants SG-11V2 (SEQ ID NO:13;comprising G84D, C147V, C151S), SG-11V3 (SEQ ID NO:15; comprising N83S,G84D, C147V, C151S), SG-11V4 (SEQ ID NO:17; comprising N53S, G84D,C147V, C151S) and SG-11V5 (SEQ ID NO:19; comprising N53S, N83S, C147V,C151S).

Example 10 shows that PTMs (methionine oxidation and asparaginedeamidation) is significantly reduced in SG-11V5 as compared to SG-11(SEQ ID NO:7). The reductions were observed both at differenttemperatures and in different storage buffers. Example 11 describes anexperiment performed to determine if an SG-11 variant comprising thecysteine substitutions (SG-11V5, SEQ ID NO:19) would affect aggregationof the protein in a storage buffer. The results show that the SG-11V5variant has reduced aggregation compared to SG-11 (SEQ ID NO:7) whentested in different storage buffers.

Notably, although the amino acids substituted to generate SG-11V5 arepresent in a relatively conserved region of the SG-11 protein, it waspossible to substitute these 4 residues without losing functionalactivity (Examples 12 and 13, described in more detail below).

Based on the experimental data and analysis described herein, variantsof SG-11 (e.g., SEQ ID NO:3 or SEQ ID NO:5) were designed to substituteany one or more of amino acids N53, N83, C147 and C151 of SEQ ID NO:7(wherein noted substitutions are at residue positions with respect toSEQ ID NO:7). An embodiment of this variant is provided below in Table3, as SEQ ID NO:33, wherein the residue at each of positions 53, 83, 84,147 and 151 is denoted as X indicating that one or more of these 4residues can each be substituted for any of the other 19 amino acids. Insome embodiments, the protein comprises the amino acid sequence of SEQID NO:33. In other embodiments, X53 is N, S, T, M, R, Q; and/or X83 isN, R or K; and/or X84 is G or A; and/or X147 is C, S, T, M, V, L, A, orG; and/or X151 is C, S, T, M, V, L, A, or G. In still other embodiments,X53 is N, S or K; and/or X83 is N or R; and/or X84 is G or A; and/orX147 is C, V, L or A; and/or X151 is C, S, V, L or A. In still otherembodiments, X53 is any amino acid other than N, X83 is any amino acidother than N, X84 is any amino acid other than G, X147 is any amino acidother than C, and/or X151 is any amino acid other than C.

TABLE 3 Amino Acid Sequence for SEQ ID NO: 33MLEGEESVVYVGKKGVIASLDVETLDQSYYDETELKSYVDAEVEDYTAEHG K XAVKVESLKVEDGVAKLKMKYKTPEDYTAF XX IELYQGKVVASLAAGYVYDGEFARVEEGKVVGAATKQDIYSEDDLKVAIIRANTDVKVDGEI X YVS X QNVKLTGKDSVSIRDGYYLETGSVTASVDVTGQESVGTEQLSGTEQMEMTGEPVNADDTEQTEAAAGDGSFETDVYTFIVYK

In another example, certain amino acids of the provided proteins may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, binding sites on substrate molecules, receptors,antigen-binding regions of antibodies, and the like. Thus, theseproteins would be biologically functional equivalents of the disclosedproteins (e.g., comprising SEQ ID NO:3 or variants thereof). So-called“conservative” changes do not disrupt the biological activity of theprotein, as the structural change is not one that impinges on theprotein's ability to carry out its designed function. It is thuscontemplated by the inventors that various changes may be made in thesequence of genes and proteins disclosed herein, while still fulfillingthe goals of the present disclosure.

Also described herein are variants of SG-11: SEQ ID NO:11 (C147V, C151S,“SG11-V1”), SEQ ID NO:13 (G84D, C147V, C151S “SG11-V2”), SEQ ID NO:15(N83S, C147V, C151S “SG11-V3”), SEQ ID NO:17 (N535, G84D, C147V, C151S“SG11-V4”), and SEQ ID NO:19 (N53S, N83S, C147V, C151S “SG11-V5”).

Importantly, the SG-11 variant protein comprising SEQ ID NO:19maintained its activity both with respect to the TEER assay (Example 12)and in vivo DSS mouse models (Example 13), showing that variants ofSG-11 can maintain therapeutic function equivalent to that of wild typeSG-11. Specifically, in vitro TEER and in vivo DSS model experimentswere performed in which SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19)were used in parallel. Example 12 shows that SG-11 and SG-11V5 hadessentially the same functional ability to reduce TEER in vitro. Asdescribed in Examples 4 and 5 in which DSS model mice were treated withSG-11 before or after DSS treatment, Example 13 was performed to comparein vivo efficacy of SG-11 and the SG-11 variant. Example 13 alsocompared administration to the mice with the protein before DSS(described as Example 13A) and after DSS (described as Example 13B)treatment. SG-11 and the SG-11 variant reduced weight loss (FIGS. 20Aand 20B) as well as gross pathology clinical scores (FIG. 21). Again,SG-11 reduced intestinal permeability and serum LBP levels while SG-11V5was shown to reduce intestinal permeability (FIG. 18A) and serum LBPlevels in a dose-dependent manner (FIG. 19A) in Example 13A. Similar toresults observed in Examples 4 and 5, SG-11 and the SG-11 variantprotein did not reduce intestinal permeability or serum LBP levels inExample 13B where the therapeutic protein was administered after aprolonged assault with DSS and results observed over a limited period oftime. As discussed above, it is considered that continuation of thestudy would show a decrease in both permeability and serum LBP levels.

SG-21—A Functional Fragment of SG-11

Without being bound by theory it is considered that a protein comprisingSEQ ID NO:3 or a functional variant thereof (e.g., SEQ ID NO:19) canimpart therapeutic effect when present in the lumen of the alimentarycanal, such as the mouth, small intestine and/or large intestine.Accordingly, experiments were performed to test the stability ofpurified or isolated SG-11 protein in a fecal slurry as a means ofassessing stability of the protein in the intestine. As shown in Example14 (and FIG. 24), incubation of purified SG-11 in a fecal slurryresulted in a protein having an apparent molecular weight of 25 kDa whenanalyzed by SDS-PAGE. Furthermore, digestion of purified SG-11 proteinwith trypsin, which can cleave after lysine residues, resulted in apredominant product, also with an apparent molecular weight of 25 kDA asdetermined by SDS-PAGE. The fecal slurry-treated SG-11 protein was thenshown to maintain the ability to enhance epithelial barrier functionintegrity in a TEER assay (Example 12). Peptide mapping of the apparent25 kDa band excised from an SDS-PAGE provides evidence that the 25 kDaprotein is a C-terminal portion of the SG-11 protein, herein referred toas SG-21, wherein the N-terminus is likely to be an amino acid at aposition within about residues 70 to 75, 65 to 85, or 65 to 75.

In an exemplary embodiment, a C-terminal fragment of SG-11 or variantthereof is provided which comprises residues 72 to 232 of SEQ ID NO:3 orresidues 73 to 233 of SEQ ID NO:19, wherein each of the C-terminalfragments of SEQ ID NO:3 or SEQ ID NO:19 can further comprise amethionine at the N-terminus (SEQ ID NO:36 or SEQ ID NO:42,respectively). A C-terminal fragment comprising at least a C-terminalportion of SG-11 (e.g., at least 40, 50, 75, 100, 125, 150 or 160 aminoacids of residues 50 to 232 of SEQ ID NO:3), or variant or fragmentthereof, which has functional activity equivalent to that of SG-11 isprovided herein and referred to as SG-21 or a variant or fragmentthereof. Amino acid sequences for SG-21, SEQ ID NO:34 and the SG-21V5variant SEQ ID NO:40, are provided in Table 4 below.

TABLE 4 SEQ ID NO: 34 (SG-21)YKTPEDYTAFNGIELYQGKVVASLAAGYVYDGEFARVEEGKVVGAATKQDIYSEDDLKVAIIRANTDVKVDGEICYVSCQNVKLTGKDSVSIRDGYYLETGSVTASVDVTGQESVGTEQLSGTEQMEMTGEPVNADDTEQTEAAAGDGSFETD VYTFIVYKSEQ ID NO: 40 (SG-21V5)YKTPEDYTAFSGIELYQGKVVASLAAGYVYDGEFARVEEGKVVGAATKQDIYSEDDLKVAIIRANTDVKVDGEIVYVSSQNVKLTGKDSVSIRDGYYLETGSVTASVDVTGQESVGTEQLSGTEQMEMTGEPVNADDTEQTEAAAGDGSFETD VYTFIVYK

In view of these data provided herein is a therapeutic protein is atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a proteincomprising a fragment of the SG-11 protein (e.g., SEQ ID NO:3) which isfunctionally active as demonstrated by the ability to increaseepithelial barrier function as determined by an in vitro TEER assay asdescribed herein or by the ability to improve pathology in an animalmodel of IBD such as a DSS model. For example, a functional fragment ofSG-11 is a fragment which, when administered to a mouse treated withDSS, reduces weight loss as compared to a control DSS mouse not treatedwith the fragment. A functional fragment of SG-11 as described herein isreferred to as SG-21. In some embodiments, an SG-21 protein is providedcomprising amino acids 80 to 220, 75 to 225, 75 to 232, 74 to 232, 73 to232, 72 to 232, 71 to 232, 70 to 232, 69 to 232, 68 to 232, 67 to 232 or66 to 232 of SEQ ID NO:3 or a fragment thereof. The SG-21 protein mayhave a length of about 1 to 200, 1 to 190, 1 to 180, 1 to 175, 1 to 170,1 to 165, 1 to 164, 1 to 163, 1 to 163, 1 to 161, 1 to 160, 1 to 150,150 to 180, 155 to 180, 150 to 170, 155 to 170, 150 to 165, 155 to 165,or 160 to 165 amino acids in length, In an alternative embodiment, thetherapeutic protein has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%,99.8%, 99.9%, or 100% sequence identity to SEQ ID NO:34, SEQ ID NO:36,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:46,SEQ ID NO:47 SEQ ID NO:48 or SEQ ID NO:49 or a fragment thereof. In someembodiments, the therapeutic protein comprises an amino acid sequencethat is identical to SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47 SEQ IDNO:48 or SEQ ID NO:49. The therapeutic protein alternatively can be onewhich is a variant of SEQ ID NO:3, wherein the therapeutic protein has1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to SEQID NO:34. Alternatively stated, the therapeutic protein comprises 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 non-naturally occurring amino acidsubstitutions relative to the sequence of residues 72 to 232 of SEQ IDNO:3. In some embodiments, the therapeutic protein does not comprise anamino acid sequence identical to the sequence of residues 72 to 232 ofSEQ ID NO:3.

In some embodiments, the SG-21 protein can be modified or varied by oneor more amino acid insertions or deletions. An insertion can be theaddition of 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 1 to 10, 1 to20, 1 to 30, 1 to 40 or 1 to 50) amino acids to the N-terminus and/orC-terminus of the protein and/or can be an insert of 1 or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9 or 1 to 10, 1 to 20, 1 to 30, 1 to 40 or 1 to 50)amino acids at a position located between the N- and C-terminal aminoacids. Similarly, the deletion of the 1 or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9 or 1 to 10, 1 to 20, 1 to 30, 1 to 40 or 1 to 50) amino acidscan occur at any of the N- and C-terminus and in the internal portion.

In some embodiments, a modified or variant protein is provided whichcontains at least one non-naturally occurring amino acid substitutionrelative to SEQ ID NO:3. In other embodiments, the variant proteincomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsrelative to SEQ ID NO:3. In further embodiments, the variant proteincontains the amino acid sequence as depicted in SEQ ID NO:38 (SG-21V1),SEQ ID NO:39 (SG-21V2), or SEQ ID NO:40 (SG-21V5).

In some embodiments, a therapeutic protein according to the presentdisclosure encompasses any one of the variant proteins (e.g., SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:46, SEQ IDNO:47 SEQ ID NO:48 or SEQ ID NO:49) that also retains one or moreactivities of the full length mature protein depicted in, for example,SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:19 or of the SG-21 protein, forexample, SEQ ID NO:34 or SEQ ID NO:36.

An embodiment of this variant is provided below in Table 5, as SEQ IDNO:50, wherein the residue at each of positions 12, 13, 76, and 80 isdenoted as X indicating that one or more of these 3 residues can each besubstituted for any of the other 19 amino acids. The X at position 1 ofSEQ ID NO:50 can be any of the 20 amino acids or is not present. In someembodiments, the protein comprises the amino acid sequence of SEQ IDNO:50. In other embodiments, X12 is N, R or K; and/or X13 is G or A;and/or X76 is C, S, T, M, V, L, A, or G; and/or X80 is C, S, T, M, V, L,A, or G. In still other embodiments, X12 is N or R; and/or X13 is G orA; and/or X76 is C, V, L or A; and/or X80 is C, S, V, L or A. In stillother embodiments, X12 is any amino acid other than N, X13 is any aminoacid other than G, X76 is any amino acid other than C, and/or X80 is anyamino acid other than C.

TABLE 5 Amino Acid Sequence for SEQ ID NO: 50 XYKTPEDYTAF XXIELYQGKVVASLAAGYVYDGEFARVEEGKVVGAATKQD IYSEDDLKVAIIRANTDVKVDGEI X YVS XQNVKLTGKDSVSIRDGYYLETGSVTASVDVTGQESVGTEQLSGTEQMEMTGEPVNADDTEQTEAAAGDGSFET DVYTFIVYK

Also envisioned are polynucleotide sequences which encodes theseproteins. It is well known to the ordinarily skilled artisan that 2polynucleotide sequences which encode a single polypeptide sequence canshare relatively low sequence identity due to the degenerative nature ofthe genetic code. For example, if every codon in the polynucleotideencoding a 161-amino acid sequence contained at least 1 substitution inits third position, that would calculate to about 67% sequence identitybetween the 2 polynucleotides. A polynucleotide of the presentdisclosure comprises a sequence that encodes a protein that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identicalto SEQ ID NO:35 or SEQ ID NO:41.

The term “SG-21 variant” as used herein can include SG-21 proteins thatare, e.g., identical to or not identical to a protein comprising thesequence of SEQ ID NO:34 and/or which are further modified such as by aPTM or fusion or linkage to a second agent, e.g., a protein or peptide.

Protein PTMs occur in vivo and can increase the functional diversity ofthe proteome by the covalent addition of functional groups or proteins,proteolytic cleavage of regulatory subunits or degradation of entireproteins. Isolated proteins prepared according to the present disclosurecan undergo 1 or more PTMs in vivo or in vitro. The type ofmodification(s) depends on host cell in which the protein is expressedand includes but is not limited to phosphorylation, glycosylation,ubiquitination, nitrosylation (e.g., S-nitrosylation), methylation,acetylation (e.g., N-acetylation), lipidation (myristoylation,N-myristoylation, S-palmitoylation, farnesylation, S-prenylation,S-palmitoylation) and proteolysis may influence almost all aspects ofnormal cell biology and pathogenesis. The isolated and/or purified SG-11proteins or variants or fragments thereof as disclosed herein maycomprise one or more the above recited post-translational modifications.

The SG-21 protein or variant or fragment thereof may be a fusion proteinin which the N- and/or C-terminal domain is fused to a second proteinvia a peptide bond. Commonly used fusion partners well known to theordinarily skilled artisan include but are not limited to human serumalbumin and the crystallizable fragment, or constant domain of IgG, Fc.In some embodiments, the SG-21 protein or variant or fragment thereof islinked to a second protein or peptide via a disulfide bond, wherein thesecond protein or peptide comprises a cysteine residue.

As aforementioned, modifications and/or changes (e.g., substitutions,insertions, deletions) may be made in the structure of proteinsdisclosed herein. Thus, the present disclosure contemplates variation insequence of these proteins, and nucleic acids coding therefore, wherethey are nonetheless able to retain substantial activity with respect tothe functional activities assessed in various in vitro and in vivoassays as well as in therapeutic aspects of the present disclosure. Interms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity.

In some embodiments, the SG-21 protein as described herein can becharacterized by its ability to increase epithelial barrier functionintegrity as assessed by an in vitro TEER assay. The TEER assay cancomprise a layer of colonic epithelial cells consisting of a mixture ofenterocytes and goblet cells which are cultured until the cells obtaintight junction formation and barrier function capacity as assessed by ameasurement of trans-epithelial electrical resistance. The protein mayincrease electrical resistance in a TEER assay by at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% as compared to the TEER assayperformed in the absence of the protein.

It is also contemplated that the SG-21 protein is one which, whenadministered to a subject, can reduce weight loss, reduce the clinicalpathology score, or reduce colon shortening in the subject. In someembodiments, the subject is a mammal which has genetically or clinicallyinduced inflammatory disorder or dysfunctional epithelial barrierfunction. Alternatively, the animal has an idiopathic gastrointestinaldisorder involving a decrease in epithelial barrier function orintestinal inflammatory disorder. In other embodiments, the mammal is ahuman, non-human primate, or a rodent. The rodent may be a mouse or rat.

A therapeutic protein according to the present disclosure is one, whenadministered to a subject (e.g., rodent, non-human primate, or human),which can improve gastrointestinal epithelial cell barrier function,induce or increase mucin gene expression (e.g., muc2 expression),increase the structural integrity and/or functionality of agastrointestinal mucous barrier (e.g., in the small intestine, largeintestine, mouth and/or esophagus), and/or reduce inflammation in thegastrointestinal tract.

In some embodiments, the protein resulting from such a substitution,insertion and/or deletion of amino acids relative to SEQ ID NO:34 or SEQID NO:36 maintains a level of functional activity which is substantiallythe same as that of a protein comprising SEQ ID NO:7 or SEQ ID NO:34(e.g., is able to increase electrical resistance in a TEER assay whereinan epithelial cell layer was disrupted by, e.g., heat-killed E. coli).The variant protein may be useful as a therapeutic for treatment orprevention of a variety of conditions, including, but not limited toinflammatory conditions and/or barrier function disorders, including,but not limited to, inflammation of the gastrointestinal (includingoral, esophageal, and intestinal) mucosa, impaired intestinal epithelialcell gap junction integrity. In some embodiments, the modified proteinhas one or more of the following effects when administered to anindividual suffering from, or predisposed to, an inflammatory conditionand/or barrier function disorder: improvement of epithelial barrierintegrity, e.g., following inflammation induced disruption; suppressionof production of at least one pro-inflammatory cytokine (e.g., TNF-αand/or IL-23) by one or more immune cell(s); induction of mucinproduction in epithelial cells; improvement of epithelial wound healing;and/or increase in epithelial cell proliferation. Moreover, the modifiedor variant protein may be used for treatment or prevention of a disorderor condition such as, but not limited to, inflammatory bowel disease,ulcerative colitis, Crohn's disease, short bowel syndrome, GI mucositis,oral mucositis, chemotherapy-induced mucositis, radiation-inducedmucositis, necrotizing enterocolitis, pouchitis, a metabolic disease,celiac disease, inflammatory bowel syndrome, or chemotherapy associatedsteatohepatitis (CASH).

As demonstrated, e.g., in Example 3, the SG-11 protein can enhanceepithelial wound healing. Accordingly, provided herein is a therapeuticprotein comprising the amino acid sequence of SEQ ID NO:34 or SEQ IDNO:40 or a variant or fragment thereof, wherein the protein can increasewound healing in an in vitro assay. In some embodiments, the protein hasa length of about 150 to 170 or 165 to 175 amino acids. Also envisionedare fragments of SG-11 ranging in length from about 30 to 70, 40 to 60,or 45 to 55 amino acids in length. Examples of such fragments includebut are not limited to SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQID NO:49, and variants thereof, wherein such fragments have activitysimilar to that of SEQ ID NO:7 and/or SEQ ID NO:19.

Methods of Treatment

The SG-21 proteins described herein including variants (e.g., amino acidsubstitutions, deletions, insertions), modifications (e.g.,glycosylation, acetylation), SG-21 fragments and fusions thereof arecontemplated for use in treating a subject diagnosed with or sufferingfrom a disorder related to inflammation within the gastrointestinaltract and/or malfunction of epithelial barrier function within thegastrointestinal tract.

Provided herein are methods for treating a subject in need thereofcomprising administering to the subject a pharmaceutical compositioncomprising a SG-21 protein or fragment or variant thereof as describedin the present disclosure. The subject can be one who has been diagnosedwith inflammatory bowel disease, ulcerative colitis, pediatric UC,Crohn's disease, pediatric Crohn's disease, short bowel syndrome,mucositis GI mucositis, oral mucositis, mucositis of the esophagus,stomach, small intestine (duodenum, jejunum, ileum), large intestine(colon), and/or rectum, chemotherapy-induced mucositis,radiation-induced mucositis, necrotizing enterocolitis, pouchitis, ametabolic disease, celiac disease, irritable bowel syndrome, orchemotherapy associated steatohepatitis (CASH) Administration of theSG-21 pharmaceutical compositions described herein may also be usefulfor wound healing applications.

Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) classically includes ulcerative colitis(UC) and Crohn's disease (CD). The pathogenesis of inflammatory boweldisease is not known. A genetic predisposition has been suggested, and ahost of environmental factors, including bacterial, viral and, perhaps,dietary antigens, can trigger an ongoing enteric inflammatory cascade.Id. IBD can cause severe diarrhea, pain, fatigue, and weight loss. IBDcan be debilitating and sometimes leads to life-threateningcomplications. Accordingly, in some embodiments, the method of treatmentas described herein is effective to reduce, prevent or eliminate any oneor more of the symptoms described above wherein the method comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising the SG-21 protein orvariant or fragment thereof. In some embodiments, the method oftreatment results in remission.

Ulcerative Colitis

Ulcerative colitis is an inflammatory bowel disease that causeslong-lasting inflammation and sores (ulcers), in the innermost lining ofyour large intestine (colon) and rectum.

Ulcerative colitis typically presents with shallow, continuousinflammation extending from the rectum proximally to include, in manypatients, the entire colon. Fistulas, fissures, abscesses andsmall-bowel involvement are absent. Patients with limited disease (e.g.,proctitis) typically have mild but frequently recurrent symptoms, whilepatients with pancolitis more commonly have severe symptoms, oftenrequiring hospitalization. Botoman et al., “Management of InflammatoryBowel Disease,” Am. Fam. Physician, Vol. 57(1):57-68 (Jan. 1, 1998)(internal citations omitted). Thus, ulcerative colitis is an IBD thatcauses long-lasting inflammation and sores (ulcers) in the innermostlining of your large intestine (colon) and rectum.

Crohn's Disease

Unlike ulcerative colitis, Crohn's disease can involve the entireintestinal tract, from the mouth to the anus, with discontinuous focalulceration, fistula formation and perianal involvement. The terminalileum is most commonly affected, usually with variable degrees ofcolonic involvement. Subsets of patients have perianal disease withfissures and fistula formation. Only 2 to 3 percent of patients withCrohn's disease have clinically significant involvement of the uppergastrointestinal tract. Botoman et al., “Management of InflammatoryBowel Disease,” Am. Fam. Physician, Vol. 57(1):57-68 (Jan. 1, 1998)(internal citations omitted). Thus, Crohn's disease is an IBD thatcauses inflammation of the lining of your digestive tract. In Crohn'sdisease, inflammation often spreads deep into affected tissues. Theinflammation can involve different areas of the digestive tract, i.e.,the large intestine, small intestine, or both. Collagenous colitis andlymphocytic colitis also are considered inflammatory bowel diseases, butare usually regarded separately from classic inflammatory bowel disease.

Clinical Parameters of Inflammatory Bowel Disease

As previously discussed, inflammatory bowel disease encompassesulcerative colitis and Crohn's disease. There are numerous scores andclinical markers known to one of skill in the art that can be utilizedto access the efficacy of the administered proteins described herein intreating these conditions.

There are two general approaches to evaluating patients with IBD. Thefirst involves the visual examination of the mucosa and relies on theobservation of signs of damage to the mucosa, in view of the fact thatIBD is manifested by the appearance of inflammation and ulcers in the GItract. Any procedure that allows an assessment of the mucosa can beused. Examples include barium enemas, x-rays, and endoscopy. Anendoscopy may be of the esophagus, stomach and duodenum(esophagogastroduodenoscopy), small intestine (enteroscopy), or largeintestine/colon (colonoscopy, sigmoidoscopy). These techniques are usedto identify areas of inflammation, ulcers and abnormal growths such aspolyps.

Scoring systems based on this visual examination of the GI tract existto determine the status and severity of IBD, and these scoring systemsare intended to ensure that uniform assessment of different patientsoccurs, despite the fact that patients may be assessed by differentmedical professionals, in diagnosis and monitoring of these diseases aswell as in clinical research evaluations. Examples of evaluations basedon visual examination of UC are discussed and compared in Daperno M. etal (J Crohns Colitis. 2011 5:484-98).

Clinical scoring systems also exist, with the same purpose. The findingson endoscopy or other examination of the mucosa can be incorporated intothese clinical scoring systems, but these scoring systems alsoincorporate data based on symptoms such as stool frequency, rectalbleeding and physician's global assessment. IBD has a variety ofsymptoms that affect quality of life, so certain of these scoringsystems also take into account a quantitative assessment of the effecton quality of life as well as the quantification of symptoms. Both UCand CD, when present in the colon, generate a similar symptom profilewhich can include diarrhea, rectal bleeding, abdominal pain, and weightloss. See, Sands, B. E., “From symptom to diagnosis: clinicaldistinctions among various forms of intestinal inflammation.”Gastroenterology, Vol. 126, pp. 1518-1532 (2004).

One example of a scoring system for UC is the Mayo scoring system(Schroeder et al., N Eng J Med, 1987, 317:1625-1629), but others existthat have less commonly been used and include the Ulcerative ColitisEndoscopic Index of Severity (UCEIS) score (Travis et al, 2012, Gut,61:535-542), Baron Score (Baron et al., 1964, BMJ, 1:89), UlcerativeColitis Colonoscopic Index of Severity (UCCIS) (Thia et al., 2011,Inflamm Bowel Dis, 17:1757-1764), Rachmilewitz Endoscopic Index(Rachmilewitz, 1989, BMJ, 298:82-86), Sutherland Index (also known asthe UC Disease Activity Index (UCDAI) scoring system; Sutherland et al.,1987, Gastroenterology, 92:1994-1998), Matts Score (Matts, 1961, QJM,30:393-407), and Blackstone Index (Blackstone, 1984, Inflammatory boweldisease. In: Blackstone Mo. (ed.) Endoscopic interpretation: normal andpathologic appearances of the gastrointestinal tract, 1984, pp.464-494). For a review, see Paine, 2014, Gastroenterol Rep 2:161-168.Accordingly, also contemplated herein is a method for treating a subjectdiagnosed with and suffering from UC, wherein the treatment comprisesadministering a SG-21 protein or variant or fragment thereof asdescribed herein and wherein the treatment results in a decrease in theUC pathology as determined by measurement of the UCEIS score, the Baronscore, the UCCIS score, the Rachmilewitz Endoscopic Index, theSutherland Index, and/or the Blackstone Index.

An example of a scoring system for CD is the Crohn's Disease ActivityIndex (CDAI) (Sands B et al 2004, N Engl J Med 350 (9): 876-85; Best, etal. (1976) Gastroenterol. 70:439-444.); most major studies use the CDAIin order to define response or remission of disease. Calculation of theCDAI score includes scoring of the number of liquid stools over 7 days,instances and severity of abdominal pain over 7 days, general well-beingover 7 days, extraintestinal complications (e.g., arthritis/arthralgia,iritis/uveitis, erythema nodosum, pyoderma gangrenosum, aphtousstomatitis, anal fissure/fistula/abscess, and/or fever>37.8° C.), use ofantidiarrheal drugs over 7 days, present of abdominal mass, hematocrit,and body weight as a ratio of ideal/observed or percentage deviationfrom standard weight. Based on the CDAI score, the CD is classified aseither asymptomatic remission (0 to 149 points), mildly to moderatelyactive CD (150 to 220 points), moderately to severely active CD (221 to450 points), or severely active fulminant disease (451 to 1000 points).In some embodiments, the method of treatment comprising administering toa patient diagnosed with CD a therapeutically effective amount of SG-21protein or variant or fragment thereof results in a decrease in adiagnostic score of CD. For example, the score may change the diagnosisfrom severely active to mildly or moderately active or to asymptomaticremission.

The Harvey-Bradshaw index is a simpler version of the CDAI whichconsists of only clinical parameters (Harvey et al., 1980, Lancet1(8178):1134-1135). The impact on quality of life is also addressed bythe Inflammatory Bowel Disease Questionnaire (IBDQ) (Irvine et al.,1994, Gastroenterology 106: 287-296). Alternative methods furtherinclude CDEIS and SES CD (see, e.g., Levesque, et al. (2015)Gastroentrol. 148:37 57). Additionally or alternatively, diagnosisincludes assessment on a histological scale. Goblet depletion score andloss of crypts score are described in Johannson, et al. (2014) Gut63:281-291. Parameters and definitions for crypt architecture distortionare described in Simmonds, et al. (2014) BMC Gastroenterol. 14:93.Distinctions between acute inflammation and chronic inflammation aredescribed, e.g., in Simmonds, supra, and Gassier (2001) Am. J. Physiol.Gastrointest. Liver Physiol. 281:G216-G228.

In some embodiments, a method of treating an IBD, e.g., UC, is providedwherein the treatment is effective in reducing the Mayo Score. The MayoScore is a combined endoscopic and clinical scale used to assess theseverity of UC and has a scale of 1-12 The Mayo Score is a composite ofsubscores for stool frequency, rectal bleeding, findings of flexibleproctosigmoidoscopy or colonoscopy, and physician's global assessment(Paine, 2014, Gastroenterol Rep 2:161-168). With respect to rectalbleeding, blood streaks seen in the stool less than half the time isassigned 1 point, blood in most stools is assigned 2 points and pureblood passed is assigned 3 points. Regarding stool frequency, a normalnumber of daily stools is assigned 0 points, 1 or 2 more stools thannormal is assigned 1 point, 3 or 4 more stools than normal is assigned 2points, and 5 or more stools than usual is assigned 3 points. Withrespect to the endoscopy component, a score of 0 indicates normal mucosaor inactive UC, a score of 1 is given for mild disease with evidence ofmild friability, reduced vascular pattern, and mucosal erythema, a scoreof 2 is given for moderate disease with friability, erosions, completeloss of vascular pattern, and significant erythema, and a score of 3 isgiven for ulceration and spontaneous bleeding (Schroeder et al., 1987, NEngl J Med, 317:1625-1629). Global assessment by a physician assigns 0points for a finding of normal, 1 point for mild colitis, 2 points formoderate colitis and 3 points for severe colitis. Accordingly, in someembodiments, a patient treated with a SG-11 therapeutic protein orvariant or fragment thereof is successfully treated when the patientexperiences a reduction in the Mayo Score by at least 1, 2 or 3 pointsin at least one of: rectal bleeding, blood streaks seen in the stool,endoscopy subscore and physician's global assessment. In someembodiments, the method of treatment comprising administering to apatient diagnosed with UC a therapeutically effective amount of SG-21protein or variant or fragment thereof results in a decrease in adiagnostic score of UC. For example, the score may change a diagnosticscore, e.g., Mayo Score, by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11points.

Pouchitis

Additionally or alternatively, the compositions comprising a SG-21therapeutic protein or variant and methods of administration asdescribed herein can be used to treat pouchitis. Pouchitis is aninflammation of the lining of a pouch that is surgically created in thetreatment of UC. Specifically, subjects having serious UC may have theirdiseased colon removed and the bowel reconnected by a procedure calledileoanal anastomosis (IPAA) or J-pouch surgery. Pouchitis cases canrecur in many patients, manifesting either as acute relapsing pouchitisor chronic, unremitting pouchitis. Accordingly, provided herein aremethods for treating pouchitis, acute pouchitis or recurrent pouchitis.

Pouchitis activity can be classified as remission (no active pouchitis),mild to moderately active (increased stool frequency, urgency, and/orinfrequent incontinance), or severely active (frequent incontinenceand/or the patient is hospitalized for dehydration). The duration ofpouchitis can be defined as acute (less than or equal to four weeks) orchronic (four weeks or more) and the pattern classified as infrequent(1-2 acute episodes), relapsing (three or fewer episodes) or continuous.The response to medical treatment can be labeled as treatment responsiveor treatment refractory, with the medication for either case beingspecified. Accordingly, in some embodiments, a method for treating asubject diagnosed with pouchitis is provided wherein treatment with apharmaceutical composition comprising SG-21 or variant or fragmentthereof results in a decrease in the severity of the pouchitis and/orresults in remission.

Mucositis and Mucosal Barriers

The mucosa of the gastrointestinal (GI) tract is a complexmicroenvironment involving an epithelial barrier, immune cells, andmicrobes. A delicate balance is maintained in the healthy colon. Luminalmicrobes are physically separated from the host immune system by abarrier consisting of epithelium and mucus. The pathogenesis of IBD,although not fully elucidated, may involve an inappropriate hostresponse to an altered commensal flora with a dysfunctional mucousbarrier. See, Boltin et al., “Mucin Function in Inflammatory BowelDisease An Update,” J. Clin. Gastroenterol., Vol. 47(2):106-111(February 2013).

Mucositis occurs when cancer treatments (particularly chemotherapy andradiation) break down the rapidly divided epithelial cells lining thegastro-intestinal tract (which goes from the mouth to the anus), leavingthe mucosal tissue open to ulceration and infection. Mucosal tissue,also known as mucosa or the mucous membrane, lines all body passagesthat communicate with the air, such as the respiratory and alimentarytracts, and have cells and associated glands that secrete mucus. Thepart of this lining that covers the mouth, called the oral mucosa, isone of the most sensitive parts of the body and is particularlyvulnerable to chemotherapy and radiation. The oral cavity is the mostcommon location for mucositis. While the oral mucosa is the mostfrequent site of mucosal toxicity and resultant mucositis, it isunderstood that mucositis can also occur along the entire alimentarytract including the esophagus, stomach, small intestine (duodenum,jejunum, ileum), large intestine (colon), and rectum. In someembodiments, a pharmaceutical composition comprising SG-21 or a variantor fragment thereof is therapeutically effective to treat mucositis ofthe mouth, esophagus, stomach, small intestine (duodenum, jejunum,ileum), large intestine (colon), and/or rectum

Oral mucositis can lead to several problems, including pain, nutritionalproblems as a result of inability to eat, and increased risk ofinfection due to open sores in the mucosa. It has a significant effecton the patient's quality of life and can be dose-limiting (i.e.,requiring a reduction in subsequent chemotherapy doses). The WorldHealth Organization has an oral toxicity scale for diagnosis of oralmucositis: Grade 1: soreness±erythema, Grade 2: erythema, ulcers;patient can swallow solid food; Grade 3: ulcers with extensive erythema;patient cannot swallow solid food; Grade 4: mucositis to the extent thatalimentation is not possible. Grade 3 and Grade 4 oral mucositis isconsidered severe mucositis. Accordingly, provided herein is a methodfor treating a subject diagnosed with oral mucositis, whereinadministration of a pharmaceutical composition comprising SG-21 or avariant or fragment thereof reduces the grade of oral toxicity by atleast 1 point of the grade scale of 1 to 4.

Epithelial Barrier Function in IBD

Studies in recent years have identified a major role of both genetic andenvironmental factors in the pathogenesis of IBD. Markus Neurath,“Cytokines in Inflammatory Bowel Disease,” Nature Reviews Immunology,Vol. 14., 329-342 (2014). A combination of these IBD risk factors seemsto initiate alterations in epithelial barrier function, thereby allowingthe translocation of luminal antigens (for example, bacterial antigensfrom the commensal microbiota) into the bowel wall. Id. Subsequently,aberrant and excessive cytokine responses to such environmental triggerscause subclinical or acute mucosal inflammation in a geneticallysusceptible host. Id. Thus, the importance of proper epithelial barrierfunction in IBD is apparent, for in patients that fail to resolve acuteintestinal inflammation, chronic intestinal inflammation develops thatis induced by the uncontrolled activation of the mucosal immune system.In particular, mucosal immune cells, such as macrophages, T cells, andthe subsets of innate lymphoid cells (ILCs), seem to respond tomicrobial products or antigens from the commensal microbiota byproducing cytokines that can promote chronic inflammation of thegastrointestinal tract. Consequently, restoring proper epithelialbarrier function to patients may be critical in resolving IBD.

Colon Shortening

Ulcerative colitis is an idiopathic inflammatory bowel disease thataffects the colonic mucosa and is clinically characterized by diarrhea,abdominal pain and hematochezia. The extent of disease is variable andmay involve only the rectum (ulcerative proctitis), the left side of thecolon to the splenic flexure, or the entire colon (pancolitis). Theseverity of the disease may also be quite variable histologically,ranging from minimal to florid ulceration and dysplasia. Carcinoma maydevelop. The typical histological (microscopic) lesion of ulcerativecolitis is the crypt abscess, in which the epithelium of the cryptbreaks down and the lumen fills with polymorphonuclear cells. The laminapropria is infiltrated with leukocytes. As the crypts are destroyed,normal mucosal architecture is lost and resultant scarring shortens andcan narrow the colon. Thus, colon shortening can be a consequence ofcolitis disease and is often used diagnostically. For example,non-invasive plain abdominal x-rays can demonstrate the gaseous outlineof the transverse colon in the acutely ill patient. Shortening of thecolon and loss of haustral markings can also be demonstrated by plainfilms, as well as a double-contrast barium enema. Indications ofulcerative disease include loss of mucosal detail, cobblestone fillingdefects, and segmental areas of involvement. See, “Ulcerative Colitis:Introduction—Johns Hopkins Medicine,” found at:www.hopkinsmedicine.org/gastroenterology_hepatology/_pdfs/small_large_intestine/ulcerative_colitis.pdf.

Further, art recognized in vivo models of colitis will utilizeshortening of colon length in scoring the severity of colitis in themodel. See, Kim et al., “Investigating Intestinal Inflammation inDSS-induced Model of IBD,” Journal of Visualized Experiments, Vol. 60,pages 2-6 (February 2012).

Epithelial Barrier Function in non-IBD Diseases

An improperly functioning epithelial barrier is increasingly implicatedin, e.g., IBDs and mucositis. Moreover, there are numerous otherdiseases that studies have shown are also caused, linked, correlated,and/or exacerbated by, an improperly functioning epithelial barrier.These diseases include: (1) metabolic diseases, including—obesity, type2 diabetes, non-alcoholic steatohepatitis (NASH), non-alcoholic fattyliver disease (NAFLD), liver disorders, and alcoholic steatohepatitis(ASH); (2) celiac disease; (3) necrotizing enterocolitis; (4) irritablebowel syndrome (IBS); (5) enteric infections (e.g., Clostridiumdifficile); (6) other gastro intestinal disorders in general; (7)interstitial cystitis; (8) neurological disorders or cognitive disorders(e.g., Alzheimer's, Parkinson's, multiple sclerosis, and autism); (9)chemotherapy associated steatohepatitis (CASH); and (10) pediatricversions of the aforementioned diseases. See, e.g.: Everard et al.,“Responses of Gut Microbiota and Glucose and Lipid Metabolism toPrebiotics in Genetic Obese and Diet-Induced Leptin-Resistant Mice,”Diabetes, Vol. 60, (November 2011), pgs. 2775-2786; Everard et al.,“Cross-talk between Akkermansia muciniphila and intestinal epitheliumcontrols diet-induced obesity,” PNAS, Vol. 110, No. 22, (May 2013), pgs.9066-9071; Cani et al., “Changes in Gut Microbiota Control MetabolicEndotoxemia-Induced Inflammation in High-Fat Diet-Induced Obesity andDiabetes in Mice,” Diabetes, Vol. 57, (June 2008), pgs. 1470-1481;Delzenne et al., “Targeting gut microbiota in obesity: effects ofprebiotics and probiotics,” Nature Reviews, Vol. 7, (November 2011),pgs. 639-646. Consequently, restoring proper epithelial barrier functionto patients may be critical in resolving the aforementioned diseasestates.

A properly functioning epithelial barrier in the lumen of the alimentarycanal, including the mouth, esophagus, stomach, small intestine, largeintestine, and rectum, is critical in controlling and maintaining themicrobiome within the gastrointestinal tract and alimentary canal. Theecosystem for the microbiome includes the environment, barriers,tissues, mucus, mucin, enzymes, nutrients, food, and communities ofmicroorganism, that reside in the gastrointestinal tract and alimentarycanal. The integrity and permeability of the intestinal mucosal barrierimpacts health in many critical ways.

A loss of integrity of the mucosal barrier in gastro-intestinaldisorders due to changes in mucin secretion may be related to hostimmune changes, luminal microbial factors, or directly acting genetic orenvironmental determinants. Thus, the disequilibrium of the mucousbarrier may be central to the pathogenesis of IBD. Boltin et al., “MucinFunction in Inflammatory Bowel Disease An Update,” J. Clin.Gastroenterol., Vol. 47(2):106-111 (February 2013).

Mucins are the primary constituent of the mucous layer lining the GItract. There are at least 21 mucin (MUC) genes known in the humangenome, encoding either secreted or membrane-bound mucins. Thepredominant mucins in the normal colorectum are MUC1, MUC2, MUC3A,MUC3B, MUC4, MUC13, and MUC17.1. MUC2 is the primary secretory,gel-forming component of intestinal mucus, produced in goblet cells.See, Boltin et al., “Mucin Function in Inflammatory Bowel Disease AnUpdate,” J. Clin. Gastroenterol., Vol. 47(2):106-111 (February 2013).Along with additional secreted mucins such as MUC1, 3A, 3B, 4, 13 and17.1, goblet cell secretion of MUC2 forms a protective barrier oncolonic epithelial cells reducing exposure to intestinal contents whichmay damage epithelial cells or prime immune responses.

The dosing regimen used for treatment depends upon the desiredtherapeutic effect, on the route of administration, and on the durationof the treatment. The dose will vary from patient to patient, dependingupon the nature and severity of disease, the patient's weight, specialdiets then being followed by a patient, concurrent medication, and otherfactors which those skilled in the art will recognize.

Generally, dosage levels of therapeutic protein between 0.0001 to 10mg/kg of body weight daily are administered to the patient, e.g.,patients suffering from inflammatory bowel disease. The dosage rangewill generally be about 0.5 mg to 100.0 g per patient per day, which maybe administered in single or multiple doses.

In some aspects, the dosage range will be about 0.5 mg to 10 g perpatient per day, or 0.5 mg to 9 g per patient per day, or 0.5 mg to 8 gper patient per day, or 0.5 mg to 7 g per patient per day, or 0.5 mg to6 g per patient per day, or 0.5 mg to 5 g per patient per day, or 0.5 mgto 4 g per patient per day, or 0.5 mg to 3 g per patient per day, or 0.5mg to 2 g per patient per day, or 0.5 mg to 1 g per patient per day.

In some aspects, the dosage range will be about 0.5 mg to 900 mg perpatient per day, or 0.5 mg to 800 mg per patient per day, or 0.5 mg to700 mg per patient per day, or 0.5 mg to 600 mg per patient per day, or0.5 mg to 500 mg per patient per day, or 0.5 mg to 400 mg per patientper day, or 0.5 mg to 300 mg per patient per day, or 0.5 mg to 200 mgper patient per day, or 0.5 mg to 100 mg per patient per day, or 0.5 mgto 50 mg per patient per day, or 0.5 mg to 40 mg per patient per day, or0.5 mg to 30 mg per patient per day, or 0.5 mg to 20 mg per patient perday, or 0.5 mg to 10 mg per patient per day, or 0.5 mg to 1 mg perpatient per day.

Combination Therapies Comprising Therapeutic Proteins

The pharmaceutical compositions provided herein comprising a therapeuticprotein may be combined with other treatment therapies and/orpharmaceutical compositions. For example, a patient suffering from aninflammatory bowel disease, may already be taking a pharmaceuticalprescribed by their doctor to treat the condition. In embodiments, thepharmaceutical compositions provided herein, are able to be administeredin conjunction with the patient's existing medicines.

For example, the therapeutic proteins provided herein may be combinedwith one or more of: an anti-diarrheal, a 5-aminosalicylic acidcompound, an anti-inflammatory agent, an antibiotic, an antibody(e.g.antibodies targeting an inflammatory cytokine, e.g.antibodiestargeting an anti-cytokine agent such as anti-TNF-α, (e.g., adalimumab,certolizumab pegol, golimumab, infliximab, V565) or anti-IL-12/IL-23(e.g., ustekinumab, risankizumab, brazikumab, ustekinumab), a JAKinhibitor (e.g., tofacitinib, PF06700841, PF06651600, filgotinib,upadacitinib), an anti-integrin agent (e.g., vedolizumab, etrolizumab),a S11³ inhibitor (e.g., etrasimod, ozanimod, amiselimod), a recombinantcell-based agent) e.g., Cx601), a steroid, a corticosteroid, animmunosuppressant (e.g., azathioprine and mercaptopurine), vitamins,and/or specialized diet.

Cancer patients undergoing chemotherapy or radiation therapy andsuffering from or at risk of developing may be administered apharmaceutical composition according to the present disclosure incombination with an agent used to treat mucositis such as oralmucositis. In some embodiments, a method of treatment comprisesadministering to a patient suffering from mucositis a combination of apharmaceutical composition comprising SG-21 or a variant or fragmentthereof and one or more second therapeutic agents selected from thegroup consisting of amifostine, benzocaine, benzydamine, ranitidine,omeprazole, capsaicin, glutamine, prostaglandin E2, Vitamin E,sucralfate, and allopurinol.

In some embodiments, a synergistic effect is achieved upon combining thedisclosed therapeutic proteins with one or more additional therapeuticagents.

In some embodiments of the methods herein, the second therapeutic agentis administered in conjunction with the SG-21 protein described herein,either simultaneously or sequentially. In some embodiments, the proteinand the second agent act synergistically for treatment or prevention ofthe disease, or condition, or symptom. In other embodiments, the proteinand the second agent act additively for treatment or prevention of thedisease, or condition, or symptom.

Pharmaceutical Compositions Comprising the SG-21 Therapeutic Protein

Pharmaceutical compositions are provided herein which comprise a SG-21protein, a variant or a fragment thereof according to the presentdisclosure or pharmaceutically acceptable salts thereof and apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is formulated for administration to thegastrointestinal lumen, including the mouth, esophagus, small intestine,large intestine, rectum and/or anus.

In some embodiments, the composition comprises one or more othersubstances which are associated with the source of the protein, forexample, cellular components from a production host cell, or substanceassociated with chemical synthesis of the protein. In other embodiments,the pharmaceutical composition is formulated to include one or moresecond active agents as described herein. Moreover, the composition maycomprise ingredients that preserve the structural and/or functionalactivity of the active agent(s) or of the composition itself. Suchingredients include but are not limited to antioxidants and variousantibacterial and antifungal agents, including but not limited toparabens (e. g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

The terms “pharmaceutical” or pharmaceutically acceptable” refers tocompositions that do not or preferably do not produce an adverse,allergic, or other untoward reaction when administered to an animal,such as, for example, a human, as appropriate. The preparation of apharmaceutical composition or additional active ingredient will be knownto those of skill in the art in light of the present disclosure, asexemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover, foranimal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by the FDA Office of Biological Standards.

The pharmaceutical compositions of the disclosure are formulatedaccording to the intended route of administration and whether it is tobe administered, e.g., in solid, liquid or aerosol form. In someembodiments, the composition can be administered rectally, but may alsobe administered topically, by injection, by infusion, orally,intrathecally, intranasally, subcutaneously, mucosally, localizedperfusion bathing target cells directly, via a catheter, via a lavage,or by other method or any combination of the foregoing as would be knownto one of ordinary skill in the art. Liquid formulations comprising atherapeutically effective amount of the protein can be administeredrectally by enema, catheter, use of a bulb syringe. A suppository is anexample of a solid dosage form formulated for rectal delivery. Ingeneral, for suppositories, traditional carriers may include, forexample, polyalkylene glycols, triglycerides or combinations thereof. Incertain embodiments, suppositories may be formed from mixturescontaining, for example, the active ingredient in the range of about0.5% to about 10%, and or about 1% to about 2%. Injectable liquidcompositions are typically based upon injectable sterile saline orphosphate-buffered saline or other injectable carriers known in the art.Other liquid compositions include suspensions and emulsions. Solidcompositions such as for oral administration may be in the form oftablets, pills, capsules (e.g., hard or soft-shelled gelatin capsules),buccal compositions, troches, elixirs, suspensions, syrups, wafers, orcombinations thereof. The active agent in such liquid and solidcompositions, i.e., a protein as described herein, is typically acomponent, being about 0.05% to 10% by weight, with the remainder beingthe injectable carrier and the like.

The pharmaceutical composition may be formulated as a controlled orsustained release composition which provide release of the activeagent(s) including the therapeutic protein of the present disclosureover an extended period of time, e.g., over 30-60 minutes, or over 1-10hours, 2-8 hours, 8-24 hours, etc. Alternatively or additionally, thecomposition is formulated for release to a specific site in the hostbody. For example, the composition may have an enteric coating toprevent release of the active agent(s) in an acidic environment such asthe stomach, allowing release only in the more neutral or basicenvironment of the small intestine, colon or rectum. Alternatively oradditionally, the composition may be formulated to provide delayedrelease in the mouth, small intestine or large intestine.

Each of the above-described formulations may contain at least onepharmaceutically acceptable excipient or carrier, depending up theintended route of administration, e.g., a solid for rectaladministration or liquid for intravenous or parenteral administration oradministration via cannula. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18^(th) Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference).

The pharmaceutical compositions for administration can be present inunit dosage forms to facilitate accurate dosing. Typical unit dosageforms include prefilled, premeasured ampules or syringes of the liquidcompositions or suppositories, pills, tablets, capsules or the like inthe case of solid compositions. In some embodiments of suchcompositions, the active agent, i.e., a protein as described herein, maybe a component (about 0.1 to 50 wt/wt %, 1 to 40 wt/wt %, 0.1 to 1 wt/wt%, or 1 to 10 wt/wt %) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

The actual dosage amount in a unit dosage form of the present disclosureadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

Protein Expression Systems and Protein Production

Provided herein are compositions and methods for producing isolatedproteins of the present disclosure as well as expression vectors whichcontain polynucleotide sequence encoding the proteins and host cellswhich harbor the expression vectors.

The proteins of the present disclosure can be prepared by routinerecombinant methods, e.g., culturing cells transformed or transfectedwith an expression vector containing a nucleic acid encoding the SG-21therapeutic protein, variant or fragment thereof. Host cells comprisingany such vector are also provided. Host cells can be prokaryotic oreukaryotic and examples of host cells include E. coli, yeast, ormammalian cells. A method for producing any of the herein describedproteins is further provided and comprises culturing host cells underconditions suitable for expression of the desired protein and recoveringthe desired protein from the cell culture. The recovered protein canthen be isolated and/or purified for use in in vitro and in vivomethods, as well as for formulation into a pharmaceutically acceptablecomposition. In some embodiments, the protein is expressed in aprokaryotic cell such as E. coli and the isolation and purification ofthe protein includes step to reduce endotoxin to levels acceptable fortherapeutic use in humans or other animals.

Expression Vectors

Provided herein are expression vectors which comprise a polynucleotidesequence which encodes a protein of the present disclosure or a variantand/or fragment thereof. Polynucleotide sequences encoding the proteinsof the disclosure can be obtained using standard recombinant techniques.Desired encoding polynucleotide sequences may be amplified from thegenomic DNA of the source bacterium, i.e., R. hominis. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologous(exogenous) polynucleotides in a host cell. Many vectors that areavailable and known in the art can be used for the purpose of thepresent disclosure. Selection of an appropriate vector will dependmainly on the size of the nucleic acids to be inserted into the vectorand the particular host cell to be transformed with the vector. Eachvector contains various components, depending on its function(amplification or expression of heterologous polynucleotide, or both)and its compatibility with the particular host cell in which it resides.The vector components generally include, but are not limited to: anorigin of replication, a selection marker gene, a promoter, a ribosomebinding site (RBS), a signal sequence, the heterologous nucleic acidinsert and a transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using a pBR322, pUC, pET or pGEX vector, a plasmidderived from an E. coli species. Such vectors contain genes encodingampicillin (Amp) and tetracycline (Tet) resistance and thus provideseasy means for identifying transformed cells. These vectors as well astheir derivatives or other microbial plasmids or bacteriophage may alsocontain, or be modified to contain, promoters which can be used by themicrobial organism for expression of endogenous proteins.

An expression vector of the present disclosure may comprise a promoter,an untranslated regulatory sequence located upstream (5′) and operablylinked to a protein-encoding nucleotide sequence such that the promoterregulated transcription of that coding sequence. Prokaryotic promoterstypically fall into two classes, inducible and constitutive. Aninducible promoter is a promoter that initiates increased levels oftranscription of the encoding polynucleotide under its control inresponse to changes in the culture condition, e.g., the presence orabsence of a nutrient or a change in temperature. A large number ofpromoters recognized by a variety of potential host cells are well knownand a skilled artisan can choose the promoter according to desiredexpression levels. Promoters suitable for use with prokaryotic hostsinclude E. coli promoters such as lac, trp, tac, trc and ara, viralpromoters recognized by E. coli such as lambda and T5 promoters, and theT7 and T7lac promoters derived from T7 bacteriophage. A host cellharboring a vector comprising a T7 promoter, e.g., is engineered toexpress a T7 polymerase. Such host cells include E. coli BL21(DE3),Lemo21(DE3), and NiCo21(DE3) cells. In some embodiments, the promoter isan inducible promoter which is under the control of chemical orenvironmental factors.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S-transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with(3-galactosidase, ubiquitin, and the like.

Suitable vectors for expression in both prokaryotic and eukaryotic hostcells are known in the art and some are further described herein.

Vectors of the present disclosure may further comprise a signal sequencewhich allows the translated recombinant protein to be recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Forprokaryotic host cells that do not recognize and process the signalsequences native to the heterologous polypeptides, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group consisting of the alkaline phosphatase, penicillinase,Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB,OmpA and MBP. Well-known signal sequences for use in eukaryoticexpression systems include but are not limited to interleukin-2, CDS,the Immunoglobulin Kappa light chain, trypsinogen, serum albumin, andprolactin.

The SG-21 proteins or variants or fragments thereof as described hereincan be expressed as a fusion protein or polypeptide. Commonly usedfusion partners include but are not limited to human serum albumin andthe crystallizable fragment, or constant domain of IgG, Fc. Thehistidine tag or FLAG tag can also be used to simplify purification ofrecombinant protein from the expression media or recombinant celllysate. The fusion partners can be fused to the N- and/or C-terminus ofthe protein of interest.

Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Numerouscell lines and cultures are available for use as a host cell, and theycan be obtained for example through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials. Cell types available for vectorreplication and/or expression include, but are not limited to, bacteria,such as E. coli (e.g., E. coli strain RR1, E. coli LE392, E. coli B, E.coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-,prototrophic, ATCC No. 273325), DH5a, JM109, and KCB, bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, various Pseudomonas species, as wellas a number of commercially available bacterial hosts such as SURE®Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Incertain embodiments, bacterial cells such as E. coli are particularlycontemplated as host cells.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Additional eukaryotic host cells include yeasts(e.g., Pichia pastoris and Saccharomyces cerevisiae) and cells derivedfrom insects (e.g., Spodoptera frugiperda or Trichoplusia ni). Many hostcells from various cell types and organisms are available and would beknown to one of skill in the art. Similarly, a viral vector may be usedin conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector. The selection of the appropriate host cell is deemed to bewithin the skill in the art.

Methods are well known for introducing recombinant DNA, i.e., anexpression vector, into a host cell so that the DNA is replicable,either as an extrachromosomal element or as a chromosomal integrant,thereby generating a host cell which harbors the expression vector ofinterest. Methods of transfection are known to the ordinarily skilledartisan, for example, by CaPO₄ and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact, 130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). Other methods forintroducing DNA into cells include nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orintroduction using polycations, e.g., polybrene, polyornithine. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology. 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

Accordingly, provided herein is a recombinant vector or expressionvector as described above and comprising a polynucleotide which encodesa SG-21 therapeutic protein sequence of interest (e.g., SEQ ID NO:35,SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, or which encodes the proteinof SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 orvariant thereof, and/or fragment thereof as described herein). Moreover,the present disclosure provides a host cell harboring the vector. Thehost cell can be a eukaryotic or prokaryotic cell as detailed above. Insome embodiments, the host cell is a prokaryotic cell. In anotherembodiment, the host cell is E. coli.

In some embodiments, the polynucleotide encoding the protein of interestis codon-optimized. A codon optimization algorithm is applied to apolynucleotide sequence encoding a protein in order to choose anappropriate codon for a given amino acid based on the expression host'scodon usage bias. Many codon optimization algorithms also take intoaccount other factors such as mRNA structure, host GC content, ribosomalentry sites. Some examples of codon optimization algorithms and genesynthesis service providers are: AU™: www.atum.bio/services/genegps;GenScript: www.genscript.com/codon-opt.html; ThermoFisher:www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html;and Integrated DNA Technologies: www.idtdna.com/CodonOpt.

Methods to Produce the Protein

Methods are provided for producing the proteins described herein but arewell known to the ordinarily skilled artisan. Host cells transformed ortransfected with expression or cloning vectors described herein forprotein production are cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting and/or maintainingtransformants, and/or expressing the genes encoding the desired proteinsequences. The culture conditions, such as media, temperature, pH andthe like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.(IRL Press, 1991) and Molecular Cloning: A Laboratory Manual (Sambrook,et al., 1989, Cold Spring Harbor Laboratory Press).

Generally, “purified” will refer to a specific protein composition thathas been subjected to fractionation to remove non-proteinaceouscomponents and various other proteins, polypeptides, or peptides, andwhich composition substantially retains its activity, as may beassessed, for example, by the protein assays, as described herein below,or as would be known to one of ordinary skill in the art for the desiredprotein, polypeptide or peptide.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific protein, polypeptide, or peptide formsthe major component of the composition, such as constituting about 50%of the proteins in the composition or more. In some embodiments, asubstantially purified protein will constitute more than 60%, 70%, 80%,90%, 95%, 99% or even more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present disclosure, means that the peptide, polypeptideor protein has a level of purity where the peptide, polypeptide orprotein is substantially free from other proteins and biologicalcomponents. For example, a purified peptide, polypeptide or protein willoften be sufficiently free of other protein components so thatdegradative sequencing may be performed successfully.

Although preferred for use in certain embodiments, there is no generalrequirement that the protein, polypeptide, or peptide always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified protein, polypeptide or peptide, which arenonetheless enriched in the desired protein compositions, relative tothe natural state, will have utility in certain embodiments.

Various methods for quantifying the degree of purification of proteins,polypeptides, or peptides will be known to those of skill in the art inlight of the present disclosure. These include, for example, determiningthe specific protein activity of a fraction, or assessing the number ofpolypeptides within a fraction by gel electrophoresis.

Another example is the purification of a specific fusion protein using aspecific binding partner. Such purification methods are routine in theart. As the present disclosure provides DNA sequences for the specificproteins, any fusion protein purification method can now be practiced.This is exemplified by the generation of a specific protein-glutathioneS-transferase fusion protein, expression in E. coli, and isolation tohomogeneity using affinity chromatography on glutathione-agarose or thegeneration of a poly-histidine tag on the N- or C-terminus of theprotein, and subsequent purification using Ni-affinity chromatography.However, given many DNA and proteins are known, or may be identified andamplified using the methods described herein, any purification methodcan now be employed.

In other embodiments, a preparation enriched with the peptides may beused instead of a purified preparation. In this document, whenever“purified” is used, “enriched” may be used also. A preparation may notonly be enriched by methods of purification, but also by theover-expression or over-production of the peptide by bacteria whencompared to wild-type. This can be accomplished using recombinantmethods, or by selecting conditions which will induce the expression ofthe peptide from the wild type cells.

Recombinantly expressed polypeptides of the present disclosure can berecovered from culture medium or from host cell lysates. The suitablepurification procedures include, for example, by fractionation on anion-exchange (anion or cation) column; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration or size exclusion chromatograph (SEC) using, for example,Sephadex G-75; and metal chelating columns to bind epitope-tagged formsof a polypeptide of the present disclosure. Various methods of proteinpurification can be employed and such methods are known in the art anddescribed for example in Deutscher, Methods in Enzymology, 182 (1990);Scopes, Protein Purification: Principles and Practice, Springer-Verlag,New York (1982). The purification step(s) selected will depend, forexample, on the nature of the production process used and the particularpolypeptide produced.

Alternative methods, which are well known in the art, can be employed toprepare a polypeptide of the present invention. For example, a sequenceencoding a polypeptide or portion thereof, can be produced by directpeptide synthesis using solid-phase techniques (see, e.g., Stewart etal., 1969, Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif.; Merrifield. J. 1963, Am. Chem. Soc., 85:2149-2154. Invitro protein synthesis can be performed using manual techniques or byautomation. Automated synthesis can be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of a polypeptide of thepresent invention or portion thereof can be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length polypeptide or portion thereof.

In some embodiments, the disclosure provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence and the polynucleotidesencoding the chimeric molecules. Examples of such chimeric moleculesinclude, but are not limited to, any of the herein describedpolypeptides fused to an epitope tag sequence, an Fc region of animmunoglobulin.

Recombinant Bacterial Delivery Systems

The present disclosure contemplates utilizing delivery systems outsideof the traditional pharmaceutical formulations that comprise a purifiedprotein. In some embodiments, the disclosure utilizes recombinantbacterial delivery systems, phage-mediated delivery systems,chitosan-DNA complexes, or AAV delivery systems.

One particular recombinant bacterial delivery system is based uponLactococcus lactis. Essentially, one may clone the gene encoding thetherapeutic protein (e.g., SEQ ID NO:36) into an expression vector, andthen transform the vector into L. lactis. Subsequently, one may thenadminister the L. lactis to a patient. See, e.g., Brat, et al., “A phase1 trial with transgenic bacteria expressing interleukin-10 in Crohn'sdisease,” Clinical Gastroenterology and Hepatology, 2006, Vol. 4, pgs.754-759 (“We treated Crohn's disease patients with genetically modifiedLactococcus lactis (LL-Thy12) in which the thymidylate synthase gene wasreplaced with a synthetic sequence encoding mature humaninterleukin-10.”); Shigemori, et al., “Oral delivery of Lactococcuslactis that secretes bioactive heme oxygenase-1 alleviates developmentof acute colitis in mice,” Microbial Cell Factories, 2015, Vol. 14:189(“Mucosal delivery of therapeutic proteins using genetically modifiedstrains of lactic acid bacteria (gmLAB) is being investigated as a newtherapeutic strategy.”); Steidler, et al., “Treatment of murine colitisby Lactococcus lactis secreting interleukin-10,” Science, 2000, Vol.289, pgs. 1352-1355 (“The cytokine interleukin-10 (IL-10) has shownpromise in clinical trials for treatment of inflammatory bowel disease(IBD). Using two mouse models, we show that the therapeutic dose ofIL-10 can be reduced by localized delivery of a bacterium geneticallyengineered to secrete the cytokine. Intragastric administration ofIL-10-secreting Lactococcus lactis caused a 50% reduction in colitis inmice treated with dextran sulfate sodium and prevented the onset ofcolitis in IL-102/2 mice. This approach may lead to better methods forcost effective and long-term management of IBD in humans.”); Hanniffy,et al., “Mucosal delivery of a pneumococcal vaccine using Lactococcuslactis affords protection against respiratory infection,” Journal ofInfectious Diseases, 2007, Vol. 195, pgs. 185-193 (“Here, we evaluatedLactococcus lactis intracellularly producing the pneumococcal surfaceprotein A (PspA) as a mucosal vaccine in conferring protection againstpneumococcal disease.”); and Vandenbroucke, et al., “Active delivery oftrefoil factors by genetically modified Lactococcus lactis prevents andheals acute colitis in mice,” Gastroenterology, 2004, Vol. 127, pgs.502-513 (“We have positively evaluated a new therapeutic approach foracute and chronic colitis that involves in situ secretion of murine TFFby orally administered L. lactis. This novel approach may lead toeffective management of acute and chronic colitis and epithelial damagein humans.”).

In another embodiment, a “synthetic bacterium” may be used to deliver anSG-11 protein or variant or fragment thereof wherein a probioticbacterium is engineered to express the SG-11 therapeutic protein (see,e.g., Durrer and Allen, 2017, PLoS One, 12:e0176286).

Phages have been genetically engineered to deliver specific DNA payloadsor to alter host specificity. Transfer methods, such as phages,plasmids, and transposons, can be used to deliver and circulateengineered DNA sequences to microbial communities, via processes such astransduction, transformation, and conjugation. For purposes of thepresent disclosure, it is sufficient to understand that an engineeredphage could be one possible delivery system for a protein of thedisclosure, by incorporating the nucleic acid encoding said protein intothe phage and utilizing the phage to deliver the nucleic acid to a hostmicrobe that would then produce the protein after having the phagedeliver the nucleic acid into its genome.

Similar to the aforementioned engineered phage approach, one couldutilize a transposon delivery system to incorporate nucleic acidsencoding a therapeutic protein into a host microbe that is resident in apatient's microbiome. See, Sheth, et al., “Manipulating bacterialcommunities by in situ microbiome engineering,” Trends in Genetics,2016, Vol. 32, Issue 4, pgs. 189-200.

The following examples are intended to illustrate, but not limit, thedisclosure.

EXAMPLES

The following experiments utilize a robust mixture of in vitroexperiments combined with in vivo models of IBD and epithelial barrierfunction disorders to demonstrate the therapeutic ability of theprovided proteins and methods.

Example 1 Expression of SG-11 and Variants Thereof

For experiments described in the examples below, a polynucleotideencoding SG-11 (SEQ ID NO:3) was obtained by PCR amplification ofgenomic DNA obtained from Roseburia hominis (A2-183; DSM 16839 typestrain; See, e.g., Duncan, S. H., Aminov, R. I., Scott, K. P., Louis,P., Stanton, T. B., Flint, H. J. (2006). Proposal of Roseburia faecissp. nov., Roseburia hominis sp. nov. and Roseburia inulinivorans sp.nov., based on isolates from human faeces. Int.J.Syst.Evol.Microbiol.Vol. 56, pgs. 2437-2441.). The encoding polynucleotide was thensubcloned into an inducible expression vector and used to transform E.coli BL21(DE3) cells for expression and purification of SG-11 orvariants thereof as detailed below, using culturing and purificationmethods which are routine in the art.

Expression of SG-11 (Comprising SEQ ID NO:3).

Expression and purification of proteins comprising the amino acidsequence of SG-11 (SEQ ID NO:5) for use in various experimentspertaining to the present disclosure is described below was achievedusing a pGEX vector system which is designed for inducible, high-levelintracellular expression of genes or gene fragments. Expression in E.coli yields tagged proteins with the GST moiety at the amino terminusand the protein of interest at the carboxyl terminus. The vector has atac promoter for chemically inducible, high-level expression and aninternal laq1^(q) gene for use in any E. coli host.

A polynucleotide comprising a nucleotide sequence encoding SG-11 (SEQ IDNO:3 from R. hominis DSM 16839) was inserted into the multiple-cloningsite (BamHI and Nod sites) of a pGEX-6P-1 (GE Healthcare Life Science,Pittsburgh, Pa.) to express SG-11 as a GST fusion protein which was thencleaved at the Precision protease site, generating SG-11 having theamino acid sequence of SEQ ID NO:5 (encoded by SEQ ID NO:6), provided inTable 6 below. This protein was expressed and purified by 2 alternatemethods. In the first, BL21(DE3) transformants were grown in LB and 100μg/ml carbenicillin and 1 μg/ml chloramphenicol at 30° C. Expression wasinduced when a culture density of 0.6 OD₆₀₀ was reached, with 0.4 mMIPTG for 4 h. Cells were harvested by centrifugation then lysed bysonication, and a soluble lysate was applied to a GST-resin column.Bound protein was washed with PBS and then purified tag-free SG-11C waseluted by adding PreScission Protease to cleave the protein C-terminalto the GST-tag.

An alternative method of expression and purification, using the samepGEX expression construct, was performed by growing the transformedBL21(DE3) cells in LB with 50 μg/ml carbenicillin at 37° C. Whencultures reached a density of 0.7 OD₆₀₀, they were chilled to 16° C. andexpression was induced with 1 mM IPTG at 16° C. for 15 h. Cells wereharvested and lysed by sonication, and a soluble lysate was applied to aGSTrap column. Bound protein was washed with HEPES buffer and thenpurified tag-free SG-11 (SEQ ID NO:5) was eluted by adding HRV3Cprotease to cleave the protein C-terminal to the GST-tag. Elutedfractions containing protein as determined by SDS-PAGE and Coomasie Bluestaining were identified and pooled, then applied to a HiTrap Q HP anionexchange column then to a Superdex 75 (26/60) preparative size exclusioncolumn (SEC) to obtain a final preparation.

TABLE 6 Amino Acid Sequence Encoding Nucleic Acid Sequence SEQ ID NO: 5SEQ ID NO: 6 GPLGSLEGEESVVYVGKK GGGCCCCTGGGATCCCTGGAGGGAGAGGAAAGTGTCGVIASLDVETLDQSYYDET GTGTACGTGGGAAAGAAAGGCGTGATAGCGTCGCTGELKSYVDAEVEDYTAEHG GATGTGGAGACGCTCGATCAGTCCTACTACGATGAGKNAVKVESLKVEDGVAKL ACGGAACTGAAGTCCTATGTGGATGCAGAGGTGGAAKMKYKTPEDYTAFNGIEL GATTACACCGCGGAGCATGGTAAAAATGCAGTCAAGYQGKVVASLAAGYVYDG GTGGAGAGCCTTAAGGTGGAAGACGGTGTGGCGAAGEFARVEEGKVVGAATKQD CTTAAGATGAAGTACAAGACACCGGAGGATTATACCIYSEDDLKVAIIRANTDVK GCATTTAATGGAATTGAACTCTATCAGGGGAAAGTCGVDGEICYVSCQNVKLTGK TTGCTTCCCTGGCGGCAGGATACGTCTACGACGGGGADSVSIRDGYYLETGSVTAS GTTCGCCCGCGTGGAGGAAGGCAAGGTTGTGGGAGCVDVTGQESVGTEQLSGTE TGCCACAAAACAGGATATTTACTCTGAGGATGATTTGQMEMTGEPVNADDTEQTE AAAGTTGCCATCATCCGTGCCAATACGGATGTGAAGAAAGDGSFETDVYTFIVYK GTGGACGGTGAGATCTGCTATGTCTCCTGTCAGAATG AAASTGAAGCTGACCGGAAAAGACAGTGTGTCGATCCGTGACGGATATTATCTTGAGACGGGAAGCGTGACGGCATCCGTGGATGTGACCGGACAGGAGAGCGTCGGGACCGAGCAGCTTTCGGGAACCGAACAGATGGAGATGACCGGGGAGCCGGTGAATGCGGATGATACCGAGCAGACAGAGGCGGCGGCCGGTGACGGTTCGTTCGAGACAGACG TATATACTTTCATTGTCTACAAAGCGGCCGCATCG

Expression and purification of the mature SG-11 protein having no signalpeptide was done using a pD451-SR vector system (AU™, Newark, Calif.).This expression vector utilizes an IPTG-inducible T7 promoter. Thepolynucleotide encoding SG-11 (SEQ ID NO:4) was codon-optimized at AU™(Newark, Calif.) to generate the codon-optimized coding sequenceprovided herein as SEQ ID NO:8. This codon-optimized coding sequence wasinserted into the pD451-SR vector and the resultant construct providesexpression of the 233-amino acid SG-11 protein provided herein as SEQ IDNO:7.

BL21(DE3) cells transformed with the construct were grown inauto-induction media, MagicMedia (ThermoFisher). The cultures wereincubated with shaking at 25° C. for 8 hours then 16° C. for up to 72 h.Cells were pelleted by centrifugation, re-suspended in 100 mM Tris-HCl,pH 8.0 containing 50 mM NaCl, 2 mg/ml lysozyme and protease inhibitor,then Triton X-100 was added to the suspension. Cells were then sonicatedand clear lysate was prepared by centrifugation for purification of theprotein by standard column chromatography techniques.

SG-11 (SEQ ID NO:7) was purified with two anion exchange columns, HiTrapQ followed by Mono Q. Fractions containing partially purified proteinsas determined by SDS-PAGE and Coomassie Blue staining were furtherpurified with Mono Q. Purification protocol for MonoQ was the same asthat for HiTrapQ. The fractions containing SG-11 were pooled anddialyzed in buffer (50 mM sodium phosphate, 150 mM NaCl and 10%glycerol). Purity and uniformity was analyzed with SDS-PAGE andanalytical SEC, Superdex 200 Increase 3.2/300, and the preparation wasassessed to have about 92.7% purity.

The pD451-SR vector system was also used to express and purify the SG-11variant SG-11V5 (SEQ ID NO:19). To generate the expression construct,the codon-optimized sequence of SG-11 (SEQ ID NO:8) was modified togenerate the polynucleotide of SEQ ID NO:20 which encodes SG-11V5 (SEQID NO:19). The SG-11V5 encoding sequence was cloned into the pD451-SRvector.

BL21(DE3) cells transformed with the construct were grown and processedfor preparation of clear lysate as described above for expression ofSG-11 (SEQ ID NO:7).

SG-11V5 protein was purified from clear lysate by HiTrap Q purification,followed by hydrophobic interaction chromatography (HIC), HiTrap ButylHP. Fractions containing SG-11V5 and determined by SDS-PAGE andCoomassie Blue staining were pooled and dialyzed in buffer in buffer (50mM sodium phosphate, 150 mM NaCl and 10% glycerol). All columnchromatography described for preparation of SG-11 (SEQ ID NO:7) andSG-11V5 (SEQ ID NO:19 was performed using AKTA protein purificationsystems (GE Healthcare Life Sciences, Pittsburgh, Pa.).

Purified proteins were quantified by densitometry using bovine serumalbumin as a reference following SDS-PAGE and Coomassie Blue staining.Endotoxin levels were measured with Endosafe® nexgen-MCS™ (CharlesRiver, Wilmington, Mass.) according to the manufacturer's instructions.Endotoxin levels of proteins used for the assays described herein werelower than 1 EU/mg.

An expression construct was generated in which a pET-28 vector (exactvector and vendor) was used to harbor and express a polynucleotidesequence encoding SG-11 (SEQ ID NO:3) with a FLAG-tag (DYKDDDDK; SEQ IDNO:32) at the N-terminus of SG-11. The full FLAG-tagged SG-11 proteinsequence is provided herein as SEQ ID NO:9 (and was encoded bycodon-optimized polynucleotide SEQ ID NO:10). Protein expression usingthis construct is under the control of the T7 promoter, which can beinduced with isopropyl β-D-1-thiogalactopyranoside (IPTG). The FLAG-tagat the N-terminus was incorporated into the construct using PCR andoligonucleotides encoding DYKDDDDK (SEQ ID NO:32). The transformed hostcells were grown in 2×YT media overnight at 37° C. The overnight culturewas then inoculated into fresh 2×YT media and incubated at 37° C. for 4hours. The 4-hour culture was then inoculated (1% inoculation) intoMagicMedia™ E. coli Expression Medium (ThermoFisher). Cells were grownat 25° C. for 8 h and then 16° C. for up to 72 h prior to harvesting bycentrifugation. The protein was expressed as a soluble form allowingrecovery from a clear lysate. The expressed protein was purified using aHiTrapQ anion exchange column followed by a Superdex 200 Increase 10/300GL SEC. Purity and uniformity was analyzed with SDS-PAGE and analyticalSEC, Superdex 200 Increase 3.2/300, and the preparation was assessed tohave about 93.3% purity.

Preparation of SG-11 Proteins for Stability Analysis

SG-11 (SEQ ID NO:7) and a variant, SG-11V5 (SEQ ID NO:19) were purifiedwith two anion exchange columns, HiTrap Q followed by Mono Q. Fractionscontaining partially purified proteins as determined by SDS-PAGE andCoomassie Blue staining were further purified with Mono Q. Purificationprotocol for MonoQ was the same as that for HiTrapQ. The fractionscontaining SG-11 were pooled and dialyzed in buffer (50 mM sodiumphosphate, 150 mM NaCl and 10% glycerol).

For SG-11V5, following HiTrap Q purification, the protein was furtherpurified with hydrophobic interaction chromatography (HIC), HiTrap ButylHP. Fractions containing SG-11V5 and determined by SDS-PAGE andCoomassie Blue staining were pooled and dialyzed in buffer in buffer (50mM sodium phosphate, 150 mM NaCl and 10% glycerol). All columnchromatography described for preparation of and was performed using AKTAprotein purification systems (GE Healthcare Life Sciences, Pittsburgh,Pa.).

Purified proteins were quantified by densitometry using bovine serumalbumin as a reference following SDS-PAGE and Coomassie Blue staining.Endotoxin levels were measured with Endosafe® nexgen-MCS™ (CharlesRiver, Wilmington, Mass.) according to the manufacturer's instructions.Endotoxin levels of proteins used for the assays described herein werelower than 1 EU/mg.

Example 2 Effect of SG-11 on Restoration of Epithelial Barrier IntegrityFollowing Inflammation Induced Disruption

The following experiment demonstrates the therapeutic ability of aprotein as disclosed herein to restore gastrointestinal epithelialbarrier integrity. The experiment is therefore a demonstration of thefunctional utility of a therapeutic such as SG-11 to treat agastrointestinal inflammatory disorder or disease involving impairedepithelial barrier integrity/function.

Assays were performed as described below in trans-well plates whereco-cultures of multiple cell types were performed utilizing a permeablemembrane to separate cells. In the apical (top) chamber, human colonicepithelial cells, consisting of a mixture of enterocytes and gobletcells, were cultured until cells obtained tight junction formation andbarrier function capacity as assessed by measurement of trans-epithelialelectrical resistance (TEER). In the basolateral chamber, monocytes werecultured separately. Epithelial cells were primed with inflammatorycytokines. The assays measured the effect of a therapeutic protein,i.e., SG-11, on epithelial barrier function, muc2 gene expression, andproduction of cytokines.

Cell culture. The HCT8 human enterocyte cell line (ATCC Cat. No.CCL-244) was maintained in RPMI-1640 medium supplemented with 10% fetalbovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 10 μg/mlgentamicin and 0.25 μg/ml amphotericin (cRPMI). HT29-MTX human gobletcells (Sigma-Aldrich (St. Louis, Mo.; Cat. No. 12040401) were maintainedin DMEM medium with 10% fetal bovine serum, 100 IU/ml penicillin, 100μg/ml streptomycin, 10 μg/ml gentamicin and 0.25 μg/ml amphotericin(cDMEM). Epithelial cells were passaged by trypsinization and were usedbetween 5 and 15 passages following thawing from liquid nitrogen stocks.U937 monocytes (ATCC Cat. No. 700928) were maintained in cRPMI medium asa suspension culture, and split by dilution as needed to maintain cellsbetween 5×10⁵ and 2×10⁶ cells/ml. U937 cells were used up to passage 18following thawing from liquid nitrogen stocks.

Epithelial cell culture. A mixture of HCT8 enterocytes and HT29-MTXgoblet cells were plated at a 9:1 ratio, respectively, in the apicalchamber of the transwell plate as described previously (Berget et al.,2017, Int J Mol Sci, 18:1573; Beduneau et al., 2014, Eur J PharmBiopharm, 87:290-298). A total of 10⁵ cells were plated in each well(9×10⁴ HCT8 cells and 1×10⁴ HT29-MTX cells per well). Epithelial cellswere trypsinized from culture flasks and viable cells determined bytrypan blue counting. The correct volumes of each cell type werecombined in a single tube and centrifuged. The cell pellet wasresuspended in cRPMI and added to the apical chamber of the transwellplate. Cells were cultured for 8 to 10 days at 37° C.+5% CO₂, and mediawas changed every 2 days.

Monocyte culture. On day 6 of epithelial cell culture 2×10⁵ cells/wellU937 monocytes were plated into a 96 well receiver plate. Cells werecultured at 37° C.+5% CO₂ and media was changed every 24 hours for fourdays.

Co-culture assay. Following 8-10 days of culture the transwell platecontaining enterocytes were treated with 10 ng/ml IFN-γ added to thebasolateral chamber of the transwell plate for 24 hours at 37° C.+5%CO₂. After 24 hours fresh cRPMI was added to the epithelial cell cultureplate. Trans-epithelial electrical resistance (TEER) readings weremeasured after the IFN-γ treatment and were used as the pre-treatmentTEER values. SG-11 was then added to the apical chamber of the transwellplate at a final concentration of 1 μg/ml (40 nM). The myosin lightchain kinase (MLCK) inhibitor peptide 18 (BioTechne, Minneapolis, Minn.)was used at 50 nM as a positive control to prevent inflammation inducedbarrier disruption (Zolotarevskky et al., 202, Gastroenterology,123:163-172). The bacterially derived molecule staurosporine was used at100 nM as a negative control to induce apoptosis and exacerbate barrierdisruption (Antonsson and Persson, 2009, Anticancer Res, 29:2893-2898).Compounds were incubated on enterocytes for 1 hour or 6 hours. Followingpre-incubation with test compounds the transwell insert containing theenterocytes was transferred on top of the receiver plate containing U937monocytes. Heat killed E. coli (HK E. coli) (bacteria heated to 80° C.for 40 minutes) was then added to both the apical and basolateralchambers and a multiplicity of infection (MOI) of 10. Transwell plateswere incubated at 37° C.+5% CO₂ for 24 hours and a post treatment TEERmeasurement was made. The TEER assays were performed with mature SG-11protein (SEQ ID NO:5 or SEQ ID NO:9).

Data analysis. Raw electrical resistance values in ohms (Q) wereconverted to ohms per square centimeter (Ωcm²) based on the surface areaof the transwell insert (0.143 cm²). To adjust for differentialresistances developing over 10 days of culture, individual well posttreatment Ωcm² readings were normalized to pre-treatment Ωcm² readings.Normalized Ωcm² values were then expressed as a percent change from themean Ωcm² values of untreated samples.

SG-11 protein was added 30 minutes (FIG. 1A) or 6 hours (FIG. 1B) priorto exposure of both epithelial cells and monocytes to heat killedEscherichia coli (HK E. coli), inducing monocytes to produceinflammatory mediators resulting in disruption of the epithelialmonolayer as indicated by a reduction in TEER. A myosin light chainkinase (MLCK) inhibitor was utilized as a control compound, which hasbeen shown to prevent barrier disruption and/or reverse barrier losstriggered by the antibacterial immune response. Staurosporine was usedas a control compound that caused epithelial cell apoptosis and/ordeath, thus resulting in a drastic decrease in TEER, which indicatesdisruption and/or loss of epithelial cell barrier integrity/function. InFIG. 1A, SG-11 increased TEER from 55.8% disruption by HK E. coli to62%. In FIG. 1B, SG-11 increased TEER from a 53.5% disruption by HK E.coli to 60.6%. The graphs in FIGS. 1A-1B represent data pooled from twoindividual experiments (n=6).

Example 3 Effects of SG-11 on Epithelial Cell Wound Healing

The following experiment demonstrates the therapeutic ability of aprotein as disclosed herein to increase gastrointestinal epithelial cellwound healing. The experiment is therefore a demonstration of thefunctional utility of the therapeutic protein SG-11 to treat agastrointestinal inflammatory disease, or disease involving impairedepithelial barrier integrity/function, where increased epithelial cellwound healing would be beneficial.

The 96 well Oris Cell Migration assay containing plugs preventing cellattachment in the center of each well was used according to themanufacturer's instructions (Platypus Technologies, Madison, Wis.).

The migration assay plates were warmed to room temperature prior to useand plugs were removed from 100% confluence wells prior to celladdition. The HCT8 enterocyte and HT29-MTX goblet cell lines were usedat a 9:1 ratio with a total of 5×10⁴ total cells added per well (4.5×10⁴HCT8 cells and 0.5×10⁴ HT29-MTX cells). Cells were incubated at 37°C.+5% CO₂ for 24 hours. Plugs were then removed from all control andsample wells. Control wells included cells treated with the diluentvehicle as the blank, 30 ng/ml epidermal growth factor (EGF) as thepositive control, and 100 nM staurosporine as the negative control, alldiluted in cRPMI. Sample wells contained SG-11 protein (SEQ ID NO:9) ata concentration of 1 μg/ml diluted in cRPMI. 100% and 0% wells werecultured in cRPMI. Treatments were added to cells and incubated at 37°C.+5% CO₂ for 48 hours. Prior to staining for viable cells, plugs wereremoved from the 0% wells. Treatment media was removed and cells werewashed in PBS containing 0.9 mM CaCl₂ and 0.5 mM MgCl₂. The greenfluorescent viability dye Calcenin AM was added to all wells at aconcentration of 0.5 μg/ml in PBS containing 0.9 mM CaCl₂ and 0.5 mMMgCl₂, incubated for 30 min at 37° C.+5% CO₂, the dye was removed andcells were washed in PBS containing 0.9 mM CaCl₂ and 0.5 mM MgCl₂ andfluorescence was measured. Relative fluorescent values from 100% wellswhere plugs were removed prior to cell plating were set as the maxeffect, and 0% wells where plugs remained in place until immediatelybefore staining were used as the baseline. Samples were normalizedbetween 100% and 0% samples and values expressed as a percent growth.

As shown in FIG. 2, a significant increase in growth was observed upontreatment with SG-11. Control compounds modulated wound healing asexpected with EGF increasing proliferation, and staurosporinesuppressing cell proliferation. The graph in FIG. 2 represents datapooled from 5 experiments (n=15). The data represent 5 independentreplicate experiments wherein SG-11 SEQ ID NO:5 was used in 1 experimentand SEQ ID NO:9 was used in 4 experiments.

Example 4 SG-11 Demonstrates Therapeutic Activity in a Concurrent DSSModel of Inflammatory Bowel Disease

Examples 4 and 5 demonstrate the ability of a protein as disclosedherein to treat inflammatory bowel disease in an in vivo model. Theexperiment is therefore a demonstration that the aforementioned in vitromodels, which described important functional and possible mechanisticmodes of action, will translate into an in vivo model system ofinflammatory bowel disease. Specifically, the mice in Examples 4 and 5were treated with dextran sodium sulfate (DSS), a chemical known toinduce intestinal epithelial damage and thereby reduce intestinalbarrier integrity and function. DSS mice are well-accepted models ofcolitis. In Example 4, mice were treated with SG-11 proteinapproximately concurrent with (6 hours prior to) administration of DSS.In Example 5, mice were treated with DSS for 6 days prior to treatmentwith SG-11 protein.

The graphs presented in Example 4 represent data pooled from 3independent experiments, each using 10 mice (n=30). The SG-11 proteinused in these experiments was the mature protein (no signal peptide)without an N-terminal tag and comprising the amino acid sequence of SEQID NO:3. For 2 experiments, the SG-11 protein consisted of SEQ ID NO:5;for the third experiment, the SG-11 protein consisted of SEQ ID NO:7.

Eight-week old C57BL/6 mice were housed 5 animals were cage and givenfood and water ad libitum for 7 days. Following the 7-day acclimationperiod, treatments were initiated concurrently with addition of 2.5% DSSto the drinking water. Preliminary tracking studies with fluorescentlylabeled bovine serum albumin following intraperitoneal (i.p.) injectionof protein demonstrated proteins reached the colon at 6 hours after i.p.delivery. Based on these results, 6 hr prior to addition of 2.5% DSS tothe drinking water mice were treated with 50 nmoles/kg SG-11 (1.3 mg/kg)or Gly2-GLP2 (0.2 mg/kg) i.p. Six hours after the initial treatment thedrinking water was changed to water containing 2.5% DSS. The mice weretreated with 2.5% dextran sodium sulfate (DSS) in their drinking waterfor 6 days. Treatments were continued with SG-11 or Gly2-GLP2 twice aday (b.i.d.) in the morning and evening (every 8 and 16 hr) with i.p.injections at 50 nmoles/kg. Fresh 2.5% DSS drinking water was preparedevery 2 days.

On day six, mice were fasted for four hours and then orally gavaged with600 mg/kg 4KDa dextran labeled with fluorescein isothiocyanate (FITC)[4KDa-FITC]. One hour after the 4KDa-FITC gavage mice were euthanized,blood was collected and FITC signal was measured in serum. A significantincrease in 4KDa-FITC dextran translocation across the epithelialbarrier was observed in untreated mice, in comparison to vehicle treatedDSS mice. Additionally, a significant reduction in 4KDa-FITC dextran wasobserved in mice receiving DSS and treated with SG-11, as compared toDSS mice treated with vehicle. The magnitude of 4KDa-FITC dextrantranslocation observed for SG-11 was similar to the positive control ofGly2-GLP2. Results are shown in FIG. 3, and are presented as mean±SEM.The graph in FIG. 3 represents data pooled from 3 independentexperiments (n=30).

SG-11 Improves Inflammation Centric Readouts of Barrier Function in aConcurrent DSS Model of Inflammatory Bowel Disease

SG-11 was also assessed for its effects on the levels of LPS bindingprotein (LBP) in the blood of the DSS animal with and without SG-11administration. LPS binding protein (LBP), which has been linked toclinical disease activity in subjects with inflammatory bowel disease,was also measured by ELISA in the serum of mice tested in the DSS modeldescribed in Example 4. A significant increase in LBP concentration wasobserved in response to DSS. Additionally, a significant reduction inLBP was observed in SG-11 treated mice given DSS as compared to DSS micetreated with vehicle. Furthermore, SG-11 had a greater impact on LBPconcentration as compared to the control peptide Gly2-GLP2, as asignificant difference between DSS mice treated with Gly2-GLP2 and DSSmice treated with SG-11 was observed. Results are shown in FIG. 4, andare presented as mean±SEM. The graph in FIG. 4 represents data pooledfrom 3 independent experiments (n=30).

SG-11 Prevents Weight Loss in a Concurrent DSS Model of InflammatoryBowel Disease

Also assessed was the therapeutic ability of a protein as disclosedherein to ameliorate weight loss in an animal suffering from aninflammatory intestinal disorder. Weight loss is a significant andpotentially dangerous side effect of inflammatory bowel disease.

Body weight was measured daily from mice included in the DSS modeldescribed in this Example. Percent change from starting weight on day 0was determined for each mouse. SG-11 administration to DSS treated micesignificantly improved body weight as compared to vehicle treated DSSmice. Weight loss in mice treated with SG-11 at day 6 was similar toweight loss observed with Gly2-GLP2. Results are shown in FIG. 5. Thegraph in FIG. 5 represents data pooled from two independent experiments(n=20).

SG-11 Significantly Reduces Gross Pathology in a DSS Model ofInflammatory Bowel Disease

Gross pathology observations were made in mice included in theconcurrent DSS model performed in this Example. SG-11 administration toDSS treated mice significantly improved gross pathology as compared tovehicle treated DSS mice. No differences in clinical scores wereobserved between mice given DSS and treated with either Gly2-GLP2 orSG-11. The scoring system used was: (0)=no gross pathology, (1)=streaksof blood visible in feces, (2)=completely bloody fecal pellets, (3)bloody fecal material visible in cecum, (4) bloody fecal material incecum and loose stool, (5)=rectal bleeding. Results are shown in FIG. 6.The graph in FIG. 6 represents data pooled from 3 independentexperiments (n=30). These data show that SG-11 is therapeuticallyeffective in improving symptoms of IBDs such as blood in the feces.

In addition, histopathology analysis was performed on proximal anddistal colon tissues from the DSS model animals. Proximal (FIG. 7A) anddistal (FIG. 10B) colon scores (range 0-4) are presented as well as thetotal score (FIG. 7C) for the colon which represents the sum of proximaland distal colon scores (scored on a scale of 0-8). LMA=Loss of mucosalarchitecture, Edema=Edema, INF=Inflammation, TMI=Transmuralinflammation, MH=Mucosal hyperplasia, DYS=Dysplasia. Graphs representdata pooled from two independent experiments, and are plotted asmean±SEM. Statistical analysis was performed by a one-way ANOVA comparedto DSS+vehicle followed by a Fisher's LSD test for multiple comparisons.

SG-11 Minimizes the Colon Shortening Effect in Response to DSS Treatment

The following experiment demonstrates the therapeutic ability of aprotein as disclosed herein to treat inflammatory bowel disease in an invivo model, by showing an ability to prevent or minimize colonshortening.

Colon length was measured in mice included in the DSS model described inExample 7. SG-11 administration to DSS treated mice prevented colonshortening elicited by DSS. A significant improvement in colon lengthwas observed with Gly2-GLP2 and Gly2-GLP2 treatment had a significantimprovement over SG-11 treatment. Results are shown in FIG. 8A.Additionally, treatment of mice exposed to DSS with either Gly-2-GLP2 orSG-11 resulted in a significant improvement in colon weight to lengthratios (FIG. 8B). The graphs in FIGS. 8A and 8B represent data pooledfrom 3 independent experiments (n=30). Data are graphed as mean±SEM andare pooled from three independent experiments (n=30). Statisticalanalysis was performed by a one-way ANOVA followed by a Fisher's LSDmultiple comparisons test.

Example 5 SG-11 Demonstrates Therapeutic Activity in a DSS Model ofInflammatory Disease

In this example, experiments were performed to study the effects ofSG-11 in the DSS mouse model when the SG-11 protein is administered tothe mice after DSS treatment for 7 days. This differs from the treatmentregimen of Example 4 above in which mice were administered SG-11 proteinshortly before treatment with DSS. This example further demonstrates thetherapeutic ability of a protein as disclosed herein to treatinflammatory bowel disease in an in vivo model and is therefore ademonstration that the aforementioned in vitro models, which describedimportant functional and possible mechanistic modes of action, willtranslate into an in vivo model system of inflammatory bowel disease.

Eight-week-old male C57BL/6 mice were housed 5 animals per cage andgiven food and water ad libitum for seven days. Following a 7-dayacclimation period, the mice were provided with drinking watercontaining 2.5% DSS for 7 days. Fresh 2.5% DSS water was prepared every2 days during the 7 day DSS administration. For this therapeutic DSSstudy, SG-11 used to treat the animals was fused at its N-terminus to aFLAG Tag (DYKDDDDK; SEQ ID NO:32).

On day 7 normal drinking water was restored and i.p. treatments of 50nmole/kg of SG-11 (1.3 mg/kg) or Gly2-GLP2 (0.2 mg/kg) were initiated.Treatments were administered twice a day (b.i.d.), with a morning andevening dose (every 8 and 16 hours) for six days.

As detailed below, results of the treatments were analyzed with respectto animal health including body weight and gross pathology,histopathology of colon tissue, assessment of barrier disruption, andlevels of LPS binding protein.

Body weight was measured daily during the morning treatment. The colontissue was then harvested and length was measured in centimeters and thetissue was weighed. Fecal material was flushed from the colon andresidual PBS removed by gently running the colon tissue through a pairof forceps. The colon tissue was then weighed and colon weight to lengthratio in mg/mm was determined. Following weight measurements proximaland distal colon tissue was banked for RNA and protein analysis and theremaining tissues was fixed in 10% neutral buffered formalin forhistopathology. Statistical analysis was performed by a one-way ANOVAcompared to DSS+vehicle for serum 4KDa-FITC translocation, serum LBPconcentrations, colon length, and colon weight to length ratio, while atwo-way ANOVA was performed for analysis of body weight. In allanalysis, a Fisher's LSD test for multiple comparisons was used. Graphsrepresent data pooled from two experiments, and are plotted as mean±SEM.

This therapeutic model measured recovery of an established DSS insult.Because untreated mice also recover following removal of DSS from thedrinking water no increase in 4KDa-FITC signal as observed following 6days of DSS treatment (FIG. 9). Furthermore, no reduction in LBP wasobserved following Gly2-GLP2 or SG-11 treatment (FIG. 10). Therefore, nochanges in barrier function readouts were observed in the therapeuticmodel of DSS.

Although no changes in barrier function readouts were observed in thetherapeutic DSS model, significant improvements in clinical parameterssuch as body weight (FIG. 11), colon length (FIG. 12A), and colon weightto length (FIG. 12B) were observed. Similar to barrier readouts, thegross pathology scoring system based on bloody feces was no longerrelevant as even DSS mice had recovered following 6 days of treatment.However, while there was no visible blood remaining in the colon, athickened colon was still observed. From gross pathology observations, areduction in the frequency of thick colons was observed with SG-11treatment (88% in DSS+vehicle and 25% in DSS+SG-11, p<0.0001 by Fisher'sExact test, data not shown).

Histopathology analysis was performed on proximal and distal colontissues from the therapeutic DSS model described above. Proximal (FIG.13A) and distal (FIG. 13B) colon scores (range 0-4) are presented and aswell as the total score for the colon which represents the sum ofproximal and distal colon scores (Range 0-8) (FIG. 13C). LMA=Loss ofmucosal architecture, Edema=Edema, INF=Inflammation, TMI=Transmuralinflammation, MH=Mucosal hyperplasia, DYS=Dysplasia. Graphs representdata pooled from two independent experiments, and are plotted asmean±SEM. Statistical analysis was performed by a one-way ANOVA comparedto DSS+vehicle followed by a Fisher's LSD test for multiple comparisons.

SG-11 and Gly2-GLP2 treatment resulted in a modest, but significant,reduction in the loss of mucosal architecture score, with no change ininflammation and transmural inflammation scores. Similar to the resultsprovided in Example 7, similar patterns of histopathology changes wereobserved with SG-11 and Gly2-GLP2, providing additional evidence thatSG-11 may target epithelial cells.

Example 6 Design of Stable and Therapeutically Active SG-11 Variants

SG-11 is a therapeutic protein derived from the commensal bacteriumRoseburia hominis. Administration of R. hominis as a probiotic in theDSS model demonstrated efficacy with improvements in intestinal barrierfunction (4KDa-FITC and LBP), body weight, and clinical score (data notshown).

Recombinant production of a therapeutic protein can also be affected bypost-translational modifications (PTMs) which may occur duringlarge-scale expression and purification as well as during long-termstorage. Such PTMs include but are not limited to oxidation ofmethionine, deamidation of asparagine and inter- and/or intra-moleculardisulfide bonds between two cysteines. Accordingly, studies wereperformed to replace residues which may affect protein stability. Thesestudies are described in Examples 6-11.

As a first step, the SG-11 amino acid sequence (SEQ ID NO:7) was alignedto similar prokaryotic proteins. The identified residues based on thesearch results can be used for the amino acid substitution for enhancingthe stability of the therapeutic protein(s).

At first, a Blast search of the GenBank non-redundant protein database(NCBI BLAST/default parameters/BLOSUM62 matrix) was performed toidentify other prokaryotic proteins that may be homologous to SG-11. Theidentified protein sequences are shown in FIG. 14 SEQ ID NO:21 is ahypothetical protein from Roseburia intestinalis (GenBank:WP_006857001.1; BLAST E value: 3e-90); SEQ ID NO:22 is a hypotheticalprotein from Roseburia sp. 831b (GenBank:WP_075679733.1; BLAST E value:4e-58); and SEQ ID NO:23 is a hypothetical protein from Roseburiainulinivorans (GenBank: WP_055301040.1; BLAST E value: le-83).

Each of SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 is a predictedmature form of the indicated protein (lacks a signal peptide) andcontains an N-terminal methionine. A multiple sequence alignment ofthese sequences with SG-11 (SEQ ID NO:7) was performed to identifyregions conserved among the proteins. The alignment is shown in FIG. 14.The alignment was used to identify residues which were most conservedamong the different proteins in order to assess the potential impact ofsubstituting a particular amino acid(s). Portions of the SG-11 aresomewhat or highly conserved in which an amino acid at a particularposition in the protein is identical in all 4 of the aligned proteins orat least in 2 (positions) or 3 (positions) of the 4 proteins. The highsequence conservation among these homologs of SG-11 suggests that SEQ IDNO:21, SEQ ID NO:22 and SEQ ID NO:23 may also possess a functionimportant in maintaining a healthy epithelial barrier.

Example 7 Post-Translational Modification (PTM) Analysis of SG-11

Studies were performed to identify residues of SG-11 particularlysusceptible to PTMs using LC/MS/MS. The analysis was performed byLakePharma (Belmont, Calif.) to 1) confirm the amino acid sequence ofSG11 (SEQ ID NO:9), and 2) determine any post-translational modificationwhich could lead to reduced biological activity and immunogenicity,particularly deamidation and oxidation.

For peptide mapping and PTM analysis, samples were treated with DTT andIAA, followed by trypsin digestion. The digested sample was thenanalyzed by Waters ACQUITY UPLC coupled to Xevo G2-XS QTOF massspectrometer using a Protein BEH C18 column.

Peptide mapping and sequencing confirmed the predicted amino acidsequence and also indicated multiple deamidation sites and one oxidationsite. Among them, 7.84% of N53 and 3.77% N83 is deamidated. Theseresults presented in Table 7 indicate that N53 and N83 are primary sitesof deamidation under non-stress conditions. N53 indicates Asparagine(Asn; N) located at the 53th position in mature SG-11 with a methionineat the first position (SEQ ID NO:7).

TABLE 7 Post-Translation Modification of SG-11 Amino SEQ ID % total %Acid¹ NO Peptide Modifiers ion² peptide³ N53 30 NAVK Deamidation 0.017.84 N83 26 TPEDYTAFNGIELYQGK Deamidation 0.25 3.77 N137 27 ANTDVKDeamidation 0.25 1.03 N153 28 VDGEICYVSCQNVK Deamidation 0.01 0.2 M1 24MLEGEESVVYVGK Oxidation <0.01 N/A ¹Amino acid position in SG-11 (SEQ IDNO: 7) ²Normalized to total peptide ion intensity ³Normalized to thetotal intensity of corresponding precursor with or without modification

Example 8 Forced Degradation of SG-11

SG-11 (SEQ ID NO:9) was also tested under a series of stress conditionsshown in Table 8 below to further characterized the stability ofrecombinant, purified SG-11. Stressed samples were analyzed either bySEC-HPLC for the presence of aggregates and/or degradants. LC/MS/MS wasperformed for determination of levels of deamidation and oxidation.

TABLE 8 Analytic Stress factor method Criteria Observation Temperature 4° C. HPLC % monomer (>90% Solution clear (2 weeks) pass) 25° C. HPLC %monomer (>90% Solution clear pass) 37° C. HPLC % monomer Solution clear40° C. uPLC % monomer Solution clear LC/MS/MS Oxidation HydroperoxideuPLC % oxidation and sites Solution clear (0.005%) LC/MS/MS 40° C., 16hr Mechanical 350 rpm shake, HPLC % monomer (>90% Solution clear stress4° C., 24 hr pass) pH pH 4 and pH 9 uPLC % deamidation sites Solutionclear LC/MS/MS Freeze and −80° C. to room HPLC % monomer Solution clearthaw (6 temperature cycles)

For this analysis, SG-11 (SEQ ID NO:9) was present at a concentration of1 mg/ml in PBS (50 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH8.0), with the exception of tests under pH 4 and pH 9. For pH 4, SG-11(SEQ ID NO:9) was prepared at a concentration of 1 mg/ml in sodiumacetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 4). For pH 9,SG-11 (SEQ ID NO:9) was prepared at a concentration of 1 mg/ml in CAPSO(3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid) buffer (50 mMCAPSO, 150 mM NaCl, pH 9).

The analysis shows that the SG-11 (SEQ ID NO:9) sample treated at 4° C.has low level of aggregates. With increasing temperature, aggregationincreased. At 37° C., major aggregation occurred. In contrast,mechanical stress and repeated freeze and thaw do not cause eitherprotein aggregation or degradation.

Three samples treated by incubation at 40° C. for two weeks, oxidation(H₂O₂) or high pH 9, respectively, were analyzed by LC/MS/MS for PTMs.As shown in Table 9 below, significant deamidation of N83 occurred aftersample treatment at 40° C. with almost 100% deamidation. Significantdeamidation of N83 (37%) and oxidation of M200 (63.9%) were observed insamples treated with hydrogen Peroxide. 7.84% of N53 was deamidatedwithout any treatment.

TABLE 9 % No Peptides Modification¹ 40° C. Oxidation pH 9 treatmentMLEGEESVVYVGK No modification 99.82 99.88 99.94 99.78 (SEQ ID NO: 24)Oxidation of M 0.18 0.12 0.06 0.22 GVIASLDVETLDQSYYDETELKNo modification 99.91 99.93 99.95 100 (SEQ ID NO: 25) Deamidation 0.090.07 0.05 0 Q30 TPEDYTAFNGIELYQGK No modification 0 63 80.9 82.57(SEQ ID NO: 26) Deamidation 99.88 37 19.1 17.43 N83 Deamidation 0.12 0 00 N83 Deamidation Q89 ANTDVK No modification 85.39 83.56 76.05 98.96(SEQ ID NO: 27) Deamidation 14.61 16.44 23.95 1.04 N137 VDGEICYVSCQNVKNo modification 44.85 63.23 8.76 99.98 (SEQ ID NO: 28) Carbamidomethy33.59 34.52 71.71 NA C147 Carbamidomethy 18.45 1.93 12.45 NA C151Deamidation 0 0 0.35 0.02 N153 Deamidation Q152 Deamidation 3.1 0.310.28 0 N153 Carbamidomethy C151 GYYLETGSVTASVDVTGQESVGTE No modification96.38 19.17 88.16 100 QLSGTEQMEMTGEPVNADDTEQT Deamidation 3.62 0 11.84 0EAAAGDGSFETDVYTFIVYK N206 (SEQ ID NO: 29) Deamidation Q152 Oxidation MOxidation M198 0 16.92 0 0 Oxidation M200 0 63.9 0 0 NAVK Deamidation NANA NA 7.84 (SEQ ID NO: 30) N53 ¹Amino acid position in SG-11 (SEQ ID NO:7)

After reduction, free cysteines were artificially carbamidomethylated byiodoacetamide to block cysteine residues from oxidation in the assays.

Example 9 Cysteine Residues and the Stability of SG-11

The stability of SG-11 (SEQ ID NO:9) was evaluated following theincubation at 37° C. for one week and at 4° C. for 3 weeks in Buffer C(100 mM sodium phosphate, pH 7.0, 0.5 M sorbitol). The stability wasassessed by monitoring the aggregation formation with analytical sizeexclusion chromatography (SEC) equilibrated with Buffer D (100 mM sodiumphosphate, pH 7.0, 10% glycerol). No noticeable change was observedafter 3 weeks storage at 4° C. compared with the freshly thawed proteinas both samples showed a single peak at 1.57 mL. However, after aone-week incubation at 37° C., the sample clearly showed aggregationpeaks at 1.29 and 1.41 mL in addition to a monomer peak at 1.57 mL,which was the smallest peak. The cause of the aggregation wasinvestigated as follows. There are two cysteine residues found in SG-11at the positions 147 and 151 (relative to SEQ ID NO:7). Ellman's reagentassay revealed the presence of free sulfhydryl groups in SG-11 (SEQ IDNO:9), which indicated Cys¹⁴⁷ and/or Cys¹⁵¹ does not form stabledisulfide bonds. As free sulfhydryl groups could cause aggregation byforming unpreferable intermolecular disulfide bonds, it was examinedwhether the presence of reducing agent, such as β-mercaptoethanol, couldprevent the aggregation. Aggregation was greatly suppressed in thepresence of 2.5% (v/v) mercaptoethanol in a buffer (50 mM sodiumphosphate, 150 mM NaCl and 10% glycerol) following the 4-days incubationat 37° C., in contrast to the aggregations that were formed withoutβ-mercaptoethanol. The results suggested that Cys¹⁴⁷ and/or Cys¹⁵¹ thatprovides free sulfhydryl groups that caused aggregation.

Example 10 Post-Translational Modification of an SG-11 Variant

Although SG-11 protein is stable at high temperature, formingaggregations at 37° C. in a week could be the problem at the downstreamprocessing stage. Deamidation of asparagine residues found by LC/MS/MSare also a risk factor. In order to improve the manufacturability of aprotein comprising SEQ ID NO:3 or variants thereof, the results ofExamples 10 to 12 were considered in the design of SG-11 variants (e.g.,SG-11V1 (SEQ ID NO:11), SG-11V2 (SEQ ID NO:13), SG-11V3 (SEQ ID NO:15),SG-11V4 (SEQ ID NO:17) and SG-11V5 (SEQ ID NO:19)) to reduce incidenceof detrimental PTMs.

Examples 13-16 describe experiments performed to characterize theeffects of amino acid substitutions on stability and function of theSG-11 variant SG-11V5 (SEQ ID NO:19, comprising N53S, N83S, C147V, C151Swith respect to SEQ ID NO:7). SG-11V5 (expressed and purified asdescribed in Example 1).

In accordance with PTMs observed when SG-11 (SEQ ID NO:9) was subjectedto stress conditions (Example 11), SG-11V5 (SEQ ID NO:19) was analyzedby LC-MS/MS for post translational modifications using the methodsdescribed in Example 11, and compared with PTMs for SG-11 (SEQ ID NO:7)

For this analysis, PTMs of wildtype SG-11 (SEQ ID NO:7) and SG-11V5 (SEQID NO:19) were compared. In the first analysis (results provided inTable 10 below), the proteins were stored at a concentration of 1 mg/mlin Buffer 1 (50 mM NaPO₄ ⁻, pH 8, 150 mM NaCl, 10% glycerol) and storedfor 2 weeks at either 4° C. or 40° C. The proteins were then treatedwith DTT and IAA, followed by trypsin digestion. The digested sampleswere then analyzed by Waters ACQUITY UPLC couples to Xevo G2-XS QTOFmass spectrometer using a Protein BEH C18 column. Analysis of theproteins by LC-MS/MS showed that the SG-11V5 protein had significantlylower percentages of oxidation of the start methionine and deamidationof N137 as compared to SG-11 at both 4° C. and 40° C.

TABLE 10 Protein PTM site Mod 4° C. 40° C. SG-11 MLEGEESVVYVGKOxidation of M1 8.9% 12.5%  (SEQ ID (SEQ ID NO: 24 NO: 7) SG-11V5MLEGEESVVYVGK Oxidation of M1 2.5% 4.7% (SEQ ID (SEQ ID NO: 24) NO: 19)SG-11 TPEDYTAFNGIELYQGK Deamidation of N83 19.4%  98.1%  (SEQ ID(SEQ ID NO: 26) NO: 7) SG-11V5 TPEDYTAFSGIELYQGK Deamidation of N83 — —(SEQ ID (SEQ ID NO: 31) NO: 19) SG-11 ANTDVK Deamidation of N137 1.0%0.9% (SEQ ID (SEQ ID NO: 27) NO: 7) SG-11V5 ANTDVK Deamidation of N1370.1% 0.3% (SEQ ID (SEQ ID NO: 27) NO: 19)

In a second analysis, the SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19)proteins were each stored at 40° C. in a variety of buffers. The resultsare provided in Table 11 below. The storage buffer used in thisexperiment was 100 mM NaPO₄ ⁻, pH7, with 10% sorbitol (+Sor) or without10% sorbitol (−Sor) and with 10% glycerol (+Gly) or without 10% glycerol(−Gly) as indicated in Table 11. As the data in Table 11 demonstrate,there was a large decrease in oxidation of the methionine in the firstposition for the SG-11V5 (SEQ ID NO:19) protein as compared to the SG-11(SEQ ID NO:7) protein in all buffer conditions. There were alsodifferences in levels of N137 deamidation for the two proteins with thepresence of at least glycerol and also the presence of both sorbitol andglycerol resulting in large decreases in N137 deamidation. These datashow that substitution of amino acids in the SG-11 protein can havesignificant beneficial effects on PTMs of the protein in a solution.

TABLE 11 −Sor +Sor −Sor +Sor Protein PTM site Modification −Gly −Gly+Gly +Gly SG-11 MLEGEESVVYVGK Oxidation of  20% 12.1%  38.6%  27.8% (SEQ ID (SEQ ID NO: 24) M1 NO: 7) SG-11V5 MLEGEESVVYVGK Oxidation of2.7% 3.3% 5.6% 5.9% (SEQ ID (SEQ ID NO: 24) M1 NO: 19) SG-11TPEDYTAFNGIELYQGK Deamidation 98.1%  93.9%  96.2%  87.4%  (SEQ ID(SEQ ID NO: 26) of N83 NO: 7) SG-11V5 TPEDYTAFSGIELYQGK Deamidation — —— — (SEQ ID (SEQ ID NO: 31) of N83 NO: 19) SG-11 ANTDVK Deamidation 0.9%2.6% 3.0% 2.5% (SEQ ID (SEQ ID NO: 27) of N137 NO: 7) SG-11V5 ANTDVKDeamidation 1.5% 3.3% 0.4% 0.1% (SEQ ID (SEQ ID NO: 27) of N137 NO: 19)

Example 11 SG-11 Variant Construction and Stability Analysis

Although SG-11 protein is very stable at high temperature, formingaggregations at 37° C. in a week could be the problem at the downstreamprocessing stage. Deamidation of asparagine residues found by LC/MS/MSare also a risk factor. In order to improve the manufacturability of aprotein comprising SEQ ID NO:3 or variants thereof, the protein depictedas SG-11 (SEQ ID NO:7) was mutated to contain the following 4substitutions: N53S, N83S, C147V and C151S. This variant with 4substitutions is designated as SG-11V5, provided herein as SEQ ID NO:19.The stability of purified SG-11 and SG-11V5 was tested in differentstorage buffer formulations. SG-11V5 (SEQ ID NO:19) has about 98.3%sequence identity to SEQ ID NO:7.

Stability Analysis of SG-11

FIG. 15 shows effects of conditions on SG-11 (SEQ ID NO:7) stability.Specifically, purified SG-11 (SEQ ID NO:7) was incubated in pH 5.2(FIGS. 15A, 15B and 15C), pH 7.0 (FIGS. 15D, 15E and 15F) and pH 8.0(FIGS. 15G, 15H and 15I). Effect of additives was also tested at the 3different pH conditions: 150 mM NaCl (FIGS. 15A, 15D and 15G); 150 mMNaCl and 100 mM arginine (FIGS. 15B, 15E and 15H); and 150 mM NaCl and0.5 M sorbitol (FIGS. 15C, 15F and 15I). Stability was analyzed byanalytical SEC. Arrow heads indicate the retention time of the monomericform.

Stability Analysis of SG-11V5

FIG. 16 shows effects of conditions on SG-11V5 (SEQ ID NO:19) stability.SG-11V5 (SEQ ID NO:19) was incubated in pH 5.2 (FIGS. 16A, 16B and 16C),pH 7.0 (FIGS. 16D, 16E and 16F) and pH 8.0 (FIGS. 16G, 16H and 16I).Effect of additives was also tested at the 3 different pH conditions:150 mM NaCl (FIGS. 16A, 16D and 16G); 150 mM NaCl and 100 mM Arg (FIGS.16B, 16E and 16H); and 150 mM NaCl and 0.5 M sorbitol (FIGS. 16C, 16Fand 16I). Stability was analyzed by analytical SEC. Arrow heads indicatethe retention time of the monomeric form.

In the presence of 100 mM arginine at pH 7.0, aggregate formation of thepurified SG-11 (SEQ ID NO:7) protein was greatly suppressed. However,some small peaks were observed at earlier retention time, whichindicated there were different forms other than the monomeric form.SG-11V5 (SEQ ID NO:19) did not show large amount of aggregation underall conditions tested in this example. Even without any additives, thediscrete monomeric peak was observed. The small aggregation peak at 1.34mL were suppressed by 100 mM arginine or 0.5 M sorbitol. The purifiedSG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19) were precipitated at pH5.2.

Elevated temperature can increase protein degradation and aggregation,while also enhancing susceptibility to deamidation. To minimizepotential liabilities associated with deamidation and aggregation, themutations N53S, N83S C147V and C151S were introduced into in SG-11.Thus, SG-11V5 showed improved stability at the pH 7.0 and pH 8.0.

Example 12 In Vitro Functional Analysis of SG-11V5

An in vitro TEER assay was performed to demonstrate that SG-11 variants,e.g., SG-11V5, maintain functionality related to maintenance ofepithelial barrier function as shown for SG-11 proteins (see, e.g.,Example 2).

Cell culture was performed as described in Example 2. Briefly, following8-10 days of culture the transwell plate containing enterocytes weretreated with 10 ng/ml IFN-γ added to the basolateral chamber of thetranswell plate for 24 hours at 37° C.+5% CO₂. After 24 hours freshcRPMI was added to the epithelial cell culture plate. Trans-epithelialelectrical resistance (TEER) readings were measured after the IFN-γtreatment and were used as the pre-treatment TEER values. SG-11 (SEQ IDNO:7) or SG-11V5 (SEQ ID NO:19) was then added to the apical chamber ofthe transwell plate at a final concentration of 1 μg/ml (40 nM). Themyosin light chain kinase (MLCK) inhibitor peptide 18 (BioTechne,Minneapolis, Minn.) was used at 50 nM as a positive control to preventinflammation induced barrier disruption (Zolotarevskky et al., 202,Gastroenterology, 123:163-172). Compounds were incubated on enterocytesfor 6 hours. Following pre-incubation with test compounds the transwellinsert containing the enterocytes was transferred on top of the receiverplate containing U937 monocytes. Heat killed E. coli (HK E. coli)(bacteria heated to 80° C. for 40 minutes) was then added to both theapical and basolateral chambers and a multiplicity of infection (MOI) of10. Transwell plates were incubated at 37° C.+5% CO₂ for 24 hours and apost treatment TEER measurement was made. SG-11 (SEQ ID NO:7) increasedTEER from 78.6% disruption by HK E. coli to 89.5% (p<0.0001), whileSG-11V5 (SEQ ID NO:19) increased to 89.1% (p<0.0001) (FIG. 17).Statistical analysis was performed using a one-way ANOVA compared to HKE. coli followed by a Fisher's LSD multiple comparison test. The graphsin FIG. 17 represent data pooled from four plates performed in twoindividual experiments (n=12).

Example 13 In Vivo Functional Analysis of SG-11V5

Next, the DSS animal model experiments performed as described above inExamples 4 and 5 were repeated to test SG-11 or SG-11V5 (SEQ ID NO:19)in parallel. In these experiments, SG-11 or SG-11V5 was administered toa mouse concurrent with the initiation of treatment with DSS (as inExample 4) or after prior DSS administration. The only difference isthat mice in Example 5 were treated with SG-11 or SG-11V5 (SEQ ID NO:19)for 4 days rather than 6 days.

Briefly, in the first DSS mouse model (Example 13A), mice were treatedon day zero with test compound intraperitoneally (i.p.) and 6 hourslater DSS treatment was initiated. Doses administered included 50nmoles/kg for SG-11 (SEQ ID NO:7) (1.3 mg/ml), and Gly2-GLP2 (0.2mg/kg), and a dose response for SG-11V5 (SEQ ID NO:19) including 16nmoles/kg (0.4 mg/ml), 50 nmoles/kg (1.3 mg/ml) and 158 nmoles/kg (4.0mg/kg). The mice were treated with 2.5% DSS in their drinking water for6 days (day zero through day 6). Therapeutic protein treatments wereadministered twice a day for the duration of the DSS exposure.

In the second experiment (Example 13B), mice were provided with drinkingwater containing 2.5% DSS for 7 days. On day 7 normal drinking water wasrestored and i.p. treatments of 50 nmole/kg of SG-11 (SEQ ID NO:7) (1.3mg/kg), SG-11V5 (SEQ ID NO:19) (1.3 mg/kg), or Gly2-GLP2 (0.2 mg/kg)were initiated. Treatments were administered twice a day (b.i.d.), witha morning and evening dose (every 8 and 16 hours) for 4 days. For boththe prophylactic and therapeutic models fresh 2.5% DSS water wasprepared every 2 days during the DSS administration.

At the end of each DSS model study, mice were fasted for 4 hours andthen orally gavaged with 600 mg/kg 4KDa dextran labeled with fluoresceinisothiocyanate (FITC) [4KDa-FITC]. One hour after the 4KDa-FITC gavagemice were euthanized, blood was collected and FITC signal was measuredin serum. For the first model a significant increase in 4KDa-FITCdextran translocation across the epithelial barrier was observed invehicle treated DSS mice as compared to untreated mice. The results areillustrated in FIG. 18A: SG-11 (SEQ ID NO:7) significantly reduced the4KDa-FITC signal (p=0.04), and in FIG. 18B: SG-11V5 (SEQ ID NO:19) alsoreduced the 4KDa-FITC signal, although the difference was not reachstatistical significance (p=0.21). Data in both graphs are plotted asmean±SEM and each figure represent data from an individual experiment(n=10 per group).

Effects of SG-11V5 on Inflammation Centric Readouts of Barrier Functionin a DSS Model of inflammatory Bowel Disease

Upon completion of the DSS models above, LBP levels were measured as aninflammation centric readout of barrier function following the protocoldetailed in Example 5. Upon completion of both DSS models (Examples 13Aand 13B) blood was collected and serum was isolated. LPS binding protein(LBP) levels were measured in serum using a commercially available ELISAKit (Enzo Life Sciences). Results are provided in FIG. 19A and FIG. 19B.A significant increase in LBP was observed in the Example 13A DSS modelin response to DSS exposure. At the 50 nmoles/kg dose SG-11 (SEQ IDNO:7) and SG-11V5 (SEQ ID NO:19) similar reductions in LBP were observedalthough neither were statistically significant. However, SG-11V5 (SEQID NO:19) treatment at a higher dose of 158 nmoles/kg resulted in asignificant reduction in LBP production (p=0.003) (FIG. 19A). In theExample 13B DSS model, exposure to DSS resulted in a significantincrease in LBP production (FIG. 19B). However, no reduction in LBP wasobserved for any of the treatments and similar effects were observed forboth SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ ID NO:19). Without being boundby theory, it is thought that the long half-life of LBP in circulation(reported to be 12-24 hours) may make it difficult to observe reductionsin systemic LBP levels in the model where the LBP response is elicited(DSS is administered) prior to initiation of treatment (Behrendt, D., J.Dembinski, A. Heep, and P. Bartmann. 2004. Lipopolysaccharide bindingprotein in preterm infants. Arch Dis Child Fetal Neonatal Ed 89:F551-554).

Effects of SG-11 and SG-11V5 on Body Weight in a DSS Model ofInflammatory Bowel Disease

Body weight was measured throughout the experimental models in bothExample 13A and Example 13B. In the Example 13A DSS model (FIG. 20A)similar trends in body weight were observed for SG-11 (SEQ ID NO:7) andSG-11V5 (SEQ ID NO:19) treatments at 50 nmoles/kg, and a significantimprovement in body weight was observed at day 6 for SG-11V5 (SEQ IDNO:19) at 158 nmoles/kg. Similar patterns were observed in thetherapeutic DSS model where SG-11 (SEQ ID NO:7) and SG-11V5 (SEQ IDNO:19) at the 50 nmoles/kg dose had similar changes in body weight withboth having statistically improved body weight changes at day 11(p<0.05). For FIG. 20A and FIG. 20B, data are graphed as mean±SEM andeach graph represent data from an individual experiment. Statisticalanalysis was performed using a two-way ANOVA as compared to theDSS+vehicle group with a Fisher's LSD multiple comparison test.

Effects of SG-11 and SG-11V5 on Gross Pathology in a DSS Model ofInflammatory Bowel Disease

Gross pathology observations of colon tissue were made as described inExample 7 above. Briefly, a scoring system based on the level of visibleblood and fecal pellet consistency was used. The scoring system usedwas: (0)=no gross pathology, (1)=streaks of blood visible in feces,(2)=completely bloody fecal pellets, (3) bloody fecal material visiblein cecum, (4) bloody fecal material in cecum and loose stool, (5)=rectalbleeding. Similar results were obtained for SG-11 (SEQ ID NO:7) andSG-11V5 (SEQ ID NO:19) at the dose of 50 nmoles/kg and a dose dependenteffect was observed for SG-11V5 (SEQ ID NO:19) with the 160 nmoles/kgdose resulting in a significant improvement (p<0.002). Data, illustratedin FIG. 21, are presented as mean±SEM and include data from anindividual experiment. Statistical analysis was performed using aone-way ANOVA followed by a Fisher's LSD multiple comparison test.

Effects of SG-11 and SG-11V5 on Colon Length in a DSS Model ofInflammatory Bowel Disease

DSS models from Example 13 were also analyzed for the effect of SG-11and SG-11 variant proteins on the colon length. Colon lengthmeasurements were made for the Example 13A (FIG. 22A) or Example 13B(FIG. 22B) DSS models. Similar results were obtained with SG-11 (SEQ IDNO:7) and SG-11V5 (SEQ ID NO:19) in both DSS models where both treatmentregimens resulted in a significant increase in the colon length.However, no dose-dependent effect on colon length was observed withSG-11V5 (SEQ ID NO:19) in the prophylactic DSS model. Data in bothgraphs are presented as mean±SEM and represent data from an individualexperiment. Statistical analysis was performed using a one-way ANOVAcompared to DSS+vehicle followed by a Fishers LSD multiple comparisontest.

Effects of SG-11 and SG-11V5 on Colon Weight-to-Length Ratios in a DSSModel of Inflammatory Bowel Disease

DSS models from Example 13 were also analyzed for the effect of SG-11and SG-11 variant proteins on the colon weight-to-length ratio. Colonweight to length ratios were similar between SG-11 (SEQ ID NO:7) andSG-11V5 (SEQ ID NO:19) in the Example 13A (FIG. 23A) and Example 13B(FIG. 23B) DSS model treatment regimens. In the Example 13A treatment,all treatments and doses significantly improved colon weight to lengthratios (p<0.05). In the Example 13B treatment regiment, SG-11 (SEQ IDNO:7) and SG-11V5 (SEQ ID NO:19) both significantly improved colonweight to length ratios (p<0.01), while the positive control Gly2-GLP2did not. Statistical analysis was performed by a one-way ANOVA ascompared to DSS+vehicle using a Fisher's LSD multiple comparisons test.Data are graphed as mean±SEM and each figure represent data from asingle experiment.

Example 14

Identification of a SG-11 Variant with Lower Apparent Molecular Weight

Studies were done in order to assess stability of the SG-11 protein inthe intestinal environment, specifically, in the large intestine wherefecal matter is present. These studies are an important aspect ofdesigning a product which can be successfully delivered via rectaladministration. These studies also help to identify functional domainsof the protein. Initial studies showed that incubation of purifiedrecombinantly expressed SG-11 (these experiments were repeated withproteins depicted by SEQ ID NO:9, SEQ ID NO:7, and SEQ ID NO:19) in afecal slurry at room temperature degraded to form a predominant formwith an apparent molecular weight of about 25 kDa when analyzed bySDS-PAGE gel (4-20% Mini-PROTEAS® TGX™ precast protein gel; BioRad) andCoomassie blue staining. FIG. 24 shows results of an experiment in whichpurified SG-11 (SEQ ID NO:9) was incubated in the presence or absence offecal slurry or incubated in fecal slurry for different periods of timeat 37° C. Fecal slurry is prepared by dissolving 2 g fecal pellets(human) in 1 ml PBS buffer, in which the SG-11 protein was incubated(Lane 3: 20 μg in 20 μl reaction mix; Lanes 6-9: 60 μg in 20 μl reactionmix). Reactions were terminated by immediate transfer to sample bufferand boiling at 95° C. for 5 min. FIG. 24, Lane 1: Molecular weightmarkers (Precision Plus Protein™ Dual Color Standards (BioRad, Hercules,Calif.); Lane 2: purified SG-11 (SEQ ID NO:9); Lane 3: fecal slurryonly; Lane 4: SG-11 in fecal slurry, 10 min at 37° C.; Lane 5: fecalslurry only, 10 min at 37° C.; Lanes 6-9: SG-11 in fecal slurry for 10min, 30 min, 1 hr, 2 hr, respectively. The results show the generationof a predominant band with an apparent molecular weight of about 25 kDawith minor bands apparent by Coomassie Blue staining at 18 kDa and 10kDa.

An experiment was performed to assess generation of the fragment uponincubation in the presence of trypsin. Columns were prepared to contain100 μl immobilized Trypsin slurry, washed twice with PBS, loaded withSG-11 (SEQ ID NO:9) diluted in PBS, pH 7.4, then incubated at roomtemperature for varied times. To stop the reaction, each column wascentrifuged to remove protein from the column, then analyzed on anSDS-PAGE gel using Coomassie Blue visualization. The gel analysis isshown in FIG. 25. Lane 1: Molecular weight markers (kDa) (Precision PlusProtein™ Dual Color Standards, BioRad, Hercules, Calif.); Lane 2: SG-11(SEQ ID NO:9) only; Lanes 3-6: incubation of SG-11 with trypsin at roomtemperature for 10 min, 30 min, 1 hr, or 2 hr, respectively. These datashow that a predominant band is generated in the presence of trypsinwhich migrates to a position which appears to be the same as that of theproduct generated when SG-11 is incubated in fecal slurry, supportingthe assertion that the predominant band which migrates to an apparentmolecular weight of about 25 kDa results from cleavage of the matureSG-11 protein.

Next, SG-11 protein was incubated in fecal slurry in the absence orpresence of a trypsin inhibitor (soybean trypsin inhibitor (SBTI),Millipore Sigma, St. Louis, Mo.). SG-11 (SEQ ID NO:7) was mixed withfecal slurry as described above. The SG-11 samples were then incubatedat 37° C. for a about 1 hr prior to mixing the sample with SDS samplebuffer to terminate any further enzyme activity. Samples were thenanalyzed using SDS-PAGE (4-20% Mini-PROTEAS® TGX™ precast protein gel;BioRad) and stained with Coomasie Blue. As shown in FIG. 26, in thepresence of fecal slurry, a band appears with an apparent molecularweight of about 25 kDa. In the presence of both fecal slurry and typsininhibitor, most of the SG-11 protein remains intact. (FIG. 26: Lane 1:Molecular weight markers (kDa) (Precision Plus Protein™ Dual ColorStandards, BioRad, Hercules, Calif.); Lane 2: SG-11 (SEQ ID NO:7) inPBS; Lane 3: fecal slurry only; Lane 4: SG-11 with in fecal slurry; Lane5: SG-11 with fecal slurry and 1 μg SBTI; Lane 6: 1 μg SBTI inhibitoronly. These data show that the generation of the predominant band (whichmigrates to about 25 kDa) in fecal slurry is almost completely inhibitedin the presence of the trypsin inhibitor, supporting the assertion thatthe predominant band which migrates to an apparent molecular weight ofabout 25 kDa results from cleavage of the mature SG-11 protein.

Additional studies showed that addition of EDTA to an incubation mixturecontaining 3 μg SG-11, fecal slurry, and 1 μg SBTI resulted in thegeneration of the apparent ˜25 kDa band (data not shown).

Accordingly, it is concluded that the SG-11 protein can be processed infecal slurry in vitro and likely in vivo if exposed to intestinal fecalmatter to generate a fragment of the SG-11 protein, referred to hereinas SG-21.

Example 15 SG-21 Activity in an In Vitro Barrier Function Assay

The next study was performed to confirm that the SG-11 variant SG-21maintains functional activity equivalent to that of SG-11. Specifically,a TEER assay as described in Example 1 above, was done using a testagent comprised of fecal slurry and SG-11 protein (SEQ ID NO:9).

Mouse fecal pellets were collected from C57BL/6 mice and a fecalsuspension was prepared as described in Example 2 above. Tissue culturewas performed as described in Example 1 above. Briefly, following 8-10days of culture the transwell plate containing enterocytes were treatedwith 10 ng/ml IFN-γ added to the basolateral chamber of the transwellplate for 24 hours at 37° C.+5% CO₂. After 24 hours fresh cRPMI wasadded to the epithelial cell culture plate. Trans-epithelial electricalresistance (TEER) readings were measured after the IFN-γ treatment andwere used as the pre-treatment TEER values. Test samples included: 1μg/ml of SG-11 (SEQ ID NO:9), 1 μg/ml of SG-11 digested in the fecalslurry as described in Example 14, or an equivalent volume of fecalslurry. Treatments were added to the apical chamber of the transwellplate. The myosin light chain kinase (MLCK) inhibitor peptide 18(BioTechne, Minneapolis, Minn.) was used at 50 nM as a positive controlto prevent inflammation induced barrier disruption (Zolotarevskky etal., 2002, Gastroenterology, 123:163-172). Test and control agents wereincubated on enterocytes for 6 hours. Following pre-incubation with testand control agents, the transwell insert containing the enterocytes wastransferred on top of the receiver plate containing U937 monocytes. Heatkilled E. coli (HK E. coli) (bacteria heated to 80° C. for 40 minutes)was then added to both the apical and basolateral chambers and amultiplicity of infection (MOI) of 10. Transwell plates were incubatedat 37° C.+5% CO₂ for 24 hours and a post treatment TEER measurement wasmade. SG-11 increased TEER from 78.6% disruption by HK E. coli to 89.5%(p<0.0001), while fecal slurry-digested SG-11 increased to 90.2%(p<0.0001) (FIG. 27). Statistical analysis was performed using a one-wayANOVA compared to HK E. coli followed by a Fisher's LSD multiplecomparison test. The graph in FIG. 27 represent data pooled from fourplates performed in two individual experiments (n=12). Notably, similarresults were observed when the TEER assay was performed using SG-11 (SEQID NO:9) digested with trypsin as described in Example 14 rather thanincubated with fecal slurry (data not shown).

Example 16 Determining the SG-21 N-Terminus

The results obtained in Example 14 above indicate that SG-11 isprocessed in the intestine to a smaller fragment such as the apparent˜25 kDa fragment observed in the experiments described here.Accordingly, it was of interest to identify the portion of SG-11contained within this fragment and whether or not this fragmentpossesses functional activity comparable to the functional activity offull-length SG-11.

First, SG-11 (SEQ ID NO:9) was incubated in a fecal slurry mix or withtrypsin as above at 37° C. for about 2 hours. The reaction mixtures wererun on an SDS-PAGE and stained with Coomasie Blue as above. Individualgel slices containing the ˜25 kDa band and 2 much fainter, additionalbands (at about 18 kDa and 10 kDa) were excised and sent for peptidemapping analysis (Alphalyse Inc., Palo Alto, Calif.).

Each sample was reduced with DTT, alkylated with IAA and in-gel digestedwith trypsin. Each sample was then analyzed on a Bruker Maxis instrumentconnected with a Dionex nanoLC instrument vi an ESI-source. Equalamounts of the samples were separated by on a reversed phase using a 60min gradient program with a flow of 300 nL/min. The data were acquiredin data-dependent mode where a survey spectrum of m/z range 350-2000 isfollowed by MS/MS [m/z range 80-2000] of the most intense multiplycharged ions using collision induced dissociation. The data wereprocessed using a combination of software tools including Mascot 2.4.0,and Skyline 3.7.0.11317 to extract and match the experimental data withthe theoretical parent masses and fragmentation spectra. The data weresearched with semi-tryptic constraints and oxidation (M), pyro-glutamine(N-term Q), pyro-glutamate (N-term E) and acetylation of lysine.

Normalized peak intensities for each of 513 peptides identified byAlphalyse. From these data, total amounts of peptides having the sameamino acid start were quantified (in terms of peak height and totalarea) and mapped along the amino acid sequence. These data showed anincreased number of peptides identified starting at amino acid 73 of SEQID NO:7 (40 peptides identified) and 75 of SEQ ID NO:7 (44 peptidesidentified) for both the trypsin and the fecal digests. 28 peptides wereidentified with an N-terminus as position 71 of SEQ ID NO:7. A total of68 peptides were identified having N-termini before position 71 (havingN-termini at positions 14, 18, 36, 38, 40, 52 and 56 of SEQ ID NO:7) butthe sum of the total area and the maximum height for these peptides weresignificantly less than those of the peptides having N-termini atpositions 70 to 96 of SEQ ID NO:7. From these data, it is concluded thatthe region (between about positions 70 to 96) represent the N-terminusof the fragment which migrates to about the 25 kD position in SDS-PAGEanalysis. The C-terminal residue was not definitively identified becauseit does not contain any trypsin cleavage sites, and is therefore notdetectable by mass spectroscopy analysis.

The analysis of the peptides identified by the process above stronglysuggests that the predominant fragment observed in the SDS-PAGE analysisof the fecal-treated SG-11 protein is a C-terminal fragment of SG-2-11,e.g., comprising at least amino acids 100 of SG-11 and possibly havingan N-terminus beginning at residue 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 83, 83, 84 or 85 of SG-11 (SEQ ID NO:7).

Example 17 Expression of SG-21 and SG-21V5

To confirm that the functional activity of SG-11 resides in theC-terminal portion of the protein, expression constructs were designedand used to express a protein comprising amino acids 96-256 of SG-11 andSG-11V5.

For expression of the C-terminal fragment with an N-terminal His tag, apolynucleotide encoding amino acids 73 to 233 of SG-11 (SEQ ID NO:7) andof SG-11V5 (SEQ ID NO:19) was PCR-amplified and sub-cloned into thepET-28a vector (Novagen) using standard methods as described in Example1 above to generated proteins having the sequence disclosed herein asSEQ ID NO:44 and SEQ ID NO:45, respectively. Also expressed are SG-21and SG-21V5 proteins without N-terminal tags (SEQ ID NO:36 and SEQ IDNO:42, respectively) using standard protein expression and purificationprotocols.

Example 18 Functional Activity of SG-21 and Variants Thereof to RestoreEpithelial Barrier Integrity In Vitro

To further show that SG-21 or variants thereof possess activity which isequivalent to that of SG-11 or variants thereof, any one of the proteinsprepared as described, for example, in Example 17 above, with or withoutN-terminal tags, can be tested in in vitro TEER assays as described inExample 2 above. For example, a test protein comprising amino acids 73to 233 of SEQ ID NO:7 and having a total length of no more than 170amino acids is used in the TEER assays. The TEER assays can be performedto compare activity of the test proteins, e.g., SG-21 protein comprisingSEQ ID NO:3 with, e.g., SG-11 (SEQ ID NO:7), or to compare activity ofSG-21 protein comprising SEQ ID NO:3 with, e.g., SG-21V5 comprising SEQID NO:19 (see, e.g., Example 12 above). Additionally, an in vitro assayto measure effects of a SG-11 protein or fragment or variant thereof onepithelial barrier function, such as a TEER assay, can be used to testthe effects of SG-11 fragments such as those described herein as SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49 (see Table 12 below).

TABLE 12 Residues with SEQ respect ID  to SG-11 NO: SequenceSEQ ID NO: 36 46 YYLETGSVTASVDVTGQESVGTEQLSGTEQMEM  97-148TGEPVNADDTEQTEAAAGD 47 TPEDYTAFNGIELYQGKVVASLAAGYVYDGEFAR  4-49VEEGKVVGAATK 48 QDIYSEDDLKVAIIRANTDVKVDGEICYVSCQNVK 50-96 LTGKDSVSIRDG49 LAAGYVYDGEFARVEEGKVVGAATKQDIYSEDDL 25-74 KVAIIRANTDVKVDGE

The HCT8 human enterocyte cell line (ATCC Cat. No. CCL-244) ismaintained in RPMI-1640 medium supplemented with 10% fetal bovine serum,100 IU/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml gentamicin and0.25 μg/ml amphotericin (cRPMI). HT29-MTX human goblet cells(Sigma-Aldrich (St. Louis, Mo.; Cat. No. 12040401) are maintained inDMEM medium with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/mlstreptomycin, 10 μg/ml gentamicin and 0.25 μg/ml amphotericin (cDMEM).Epithelial cells are passaged by trypsinization and were used between 5and 15 passages following thawing from liquid nitrogen stocks. U937monocytes (ATCC Cat. No. 700928) are maintained in cRPMI medium as asuspension culture, and split by dilution as needed to maintain cellsbetween 5×10⁵ and 2×10⁶ cells/ml. U937 cells are used up to passage 18following thawing from liquid nitrogen stocks.

Epithelial cell culture. A mixture of HCT8 enterocytes and HT29-MTXgoblet cells are plated at about a 9:1 ratio, respectively, in theapical chamber of the transwell plate as described previously (Berget etal., 2017, Int J Mol Sci, 18:1573; Beduneau et al., 2014, Eur J PharmBiopharm, 87:290-298). A total of 10⁵ cells are plated in each well(9×10⁴ HCT8 cells and 1×10⁴ HT29-MTX cells per well). Epithelial cellsare trypsinized from culture flasks and viable cells determined bytrypan blue counting. The correct volumes of each cell type are combinedin a single tube and centrifuged. The cell pellet is resuspended incRPMI and added to the apical chamber of the transwell plate. Cells arecultured for 8 to 10 days at 37° C.+5% CO₂, and media is changed every 2days.

Monocyte culture. On day 6 of epithelial cell culture 2×10⁵ cells/wellU937 monocytes are plated into a 96 well receiver plate. Cells arecultured at 37° C.+5% CO₂ and media is changed every 24 hours for fourdays.

Co-culture assay. Following 8-10 days of culture the transwell platecontaining enterocytes is treated with 10 ng/ml IFN-γ added to thebasolateral chamber of the transwell plate for 24 hours at 37° C.+5%CO₂. After 24 hours fresh cRPMI is added to the epithelial cell cultureplate. Trans-epithelial electrical resistance (TEER) readings aremeasured after the IFN-γ treatment and are used as the pre-treatmentTEER values. SG-21 protein or variant thereof is then added to theapical chamber of the transwell plate at a final concentration of about1 μg/ml (40 nM). The myosin light chain kinase (MLCK) inhibitor peptide18 (BioTechne, Minneapolis, Minn.) is used at 50 nM as a positivecontrol to prevent inflammation induced barrier disruption(Zolotarevskky et al., 202, Gastroenterology, 123:163-172). Thebacterially derived molecule staurosporine is used at 100 nM as anegative control to induce apoptosis and exacerbate barrier disruption(Antonsson and Persson, 2009, Anticancer Res, 29:2893-2898). Compoundsare incubated on enterocytes for 1 hour or 6 hours. Followingpre-incubation with test compounds the transwell insert containing theenterocytes is transferred on top of the receiver plate containing U937monocytes. Heat killed E. coli (HK E. coli) (bacteria heated to 80° C.for 40 minutes) is then added to both the apical and basolateralchambers and a multiplicity of infection (MOI) of 10. Transwell platesare incubated at 37° C.+5% CO₂ for 24 hours and a post treatment TEERmeasurement is made.

Data analysis. Raw electrical resistance values in ohms (Q) can beconverted to ohms per square centimeter (Ωcm²) based on the surface areaof the transwell insert (0.143 cm²). To adjust for differentialresistances developing over 10 days of culture, individual well posttreatment Ωcm² readings can be normalized to pre-treatment Ωcm²readings. Normalized Ωcm² values are then expressed as a percent changefrom the mean Ωcm² values of untreated samples.

Test protein is added 1 hour or 6 hours prior to exposure of bothepithelial cells and monocytes to heat killed Escherichia coli (HK E.coli), inducing monocytes to produce inflammatory mediators resulting indisruption of the epithelial monolayer as indicated by a reduction inTEER. A myosin light chain kinase (MLCK) inhibitor is utilized as acontrol compound, which has been shown to prevent barrier disruptionand/or reverse barrier loss triggered by the antibacterial immuneresponse. Staurosporine is used as a control compound that causedepithelial cell apoptosis and/or death, thus resulting in a drasticdecrease in TEER, which indicates disruption and/or loss of epithelialcell barrier integrity/function.

Example 19 Functional Activity of SG-21 and Variants Thereof in an InVivo Model of Colitis

To further show that SG-21 or variants thereof possess activity which isequivalent to that of SG-11 or variants thereof, any one of the proteinsprepared as described, for example, in Example 17 above, with or withoutN-terminal tags, can be administered to an animal model of colitis asdescribed, for example, in Example 13 above. Again, a test proteincomprising amino acids 73 to 233 of SEQ ID NO:7 and having a totallength of no more than 170 amino acids is used in the in vivo assays.The in vivo assays can be performed to compare activity of the testproteins, e.g., SG-21 protein comprising SEQ ID NO:36 with, e.g., SG-11(SEQ ID NO:7), or to compare activity of SG-21 protein comprising SEQ IDNO:36 with, e.g., SG-21V5 comprising SEQ ID NO:42 (see, e.g., Examples4, 5, and 13 above).

In these experiments, for example SG-21 or SG-21V5 are administered to amouse concurrent with the initiation of treatment with DSS (as inExample 4) or after prior DSS administration. The only difference isthat mice in Example 5 were treated with SG-11 or SG-11V5 (SEQ ID NO:19)for 4 days rather than 6 days.

Briefly, in the first DSS mouse model (Example 13A), mice are treated onday zero with test compound intraperitoneally (i.p.) and 6 hours laterDSS treatment is initiated. Doses administered included 50 nmoles/kg forSG-21 (1.3 mg/ml), and Gly2-GLP2 (0.2 mg/kg), and a dose response forSG-21V5 (SEQ ID NO:19) including 16 nmoles/kg (0.4 mg/ml), 50 nmoles/kg(1.3 mg/ml) and 158 nmoles/kg (4.0 mg/kg). The mice were treated with2.5% DSS in their drinking water for 6 days (day zero through day 6).Therapeutic protein treatments were administered twice a day for theduration of the DSS exposure.

In the second experiment (Example 13B), mice are provided with drinkingwater containing 2.5% DSS for 7 days. On day 7 normal drinking water isrestored and i.p. treatments of 50 nmole/kg of SG-21 (1.3 mg/kg),SG-21V5 (1.3 mg/kg), or Gly2-GLP2 (0.2 mg/kg) are initiated. Treatmentsare administered twice a day (b.i.d.), with a morning and evening dose(every 8 and 16 hours) for 4 days. For both the prophylactic andtherapeutic models fresh 2.5% DSS water was prepared every 2 days duringthe DSS administration.

At the end of each DSS model study, mice are fasted for 4 hours and thenorally gavaged with 600 mg/kg 4KDa dextran labeled with fluoresceinisothiocyanate (FITC) [4KDa-FITC]. One hour after the 4KDa-FITC gavagemice are euthanized, blood is collected and FITC signal is measured inserum.

Effects of SG-21 and SG-21V5 on Inflammation Centric Readouts of BarrierFunction in a DSS Model of Inflammatory Bowel Disease

Upon completion of the DSS models above, LBP levels are measured as aninflammation centric readout of barrier function following the protocoldetailed in Example 4. Upon completion of both DSS models (Examples 13Aand 13B) blood is collected and serum is isolated. LPS binding protein(LBP) levels are measured in serum using a commercially available ELISAKit (Enzo Life Sciences).

Effects of SG-21 and SG-21V5 on Body Weight in a DSS Model ofInflammatory Bowel Disease

Body weight is measured throughout the experimental models in bothExample 13A and Example 13B.

Effects of SG-21 and SG-21V5 on Gross Pathology in a DSS Model ofInflammatory Bowel disease

Gross pathology observations of colon tissue are made as described inExample 4 above. Briefly, a scoring system based on the level of visibleblood and fecal pellet consistency is used. The scoring system is:(0)=no gross pathology, (1)=streaks of blood visible in feces,(2)=completely bloody fecal pellets, (3) bloody fecal material visiblein cecum, (4) bloody fecal material in cecum and loose stool, (5)=rectalbleeding.

Effects of SG-21 and SG-21V5 on Colon Length in a DSS Model ofInflammatory Bowel Disease

DSS models from Example 19 are also analyzed for the effect of SG-21 andSG-21 variant proteins on the colon length and colon weight-to-lengthratios as described in Example 13 above.

Although the foregoing disclosure has been described in some detail byway of illustration and examples, which are for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope of the disclosure, which is delineated in theappended claims. Therefore, the description should not be construed aslimiting the scope of the disclosure.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

REFERENCES

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1. A protein, comprising: an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:
 34. 2. The protein of claim 1, comprising: an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:
 34. 3. The protein of claim 1, comprising: an amino acid sequence having at least about 97% sequence identity to SEQ ID NO:
 34. 4. The protein of claim 1, comprising: an amino acid sequence having at least about 98% sequence identity to SEQ ID NO:
 34. 5. The protein of claim 1, comprising: an amino acid sequence having at least about 99% sequence identity to SEQ ID NO:
 34. 6. The protein of claim 1, comprising: the amino acid sequence of SEQ ID NO:
 34. 7. The protein of claim 1, wherein the amino acid at position 76 is valine.
 8. The protein of claim 1, wherein the amino acid at position 80 is serine.
 9. The protein of claim 1, wherein the amino acid at position 76 is valine, and the amino acid at position 80 is serine.
 10. The protein of claim 1, wherein the amino acid at position 13 is aspartic acid.
 11. The protein of claim 1, wherein the amino acid at position 13 is aspartic acid, and the amino acid at position 76 is valine, and the amino acid at position 80 is serine.
 12. The protein of claim 1, wherein the amino acid at position 12 is serine.
 13. The protein of claim 1, wherein the amino acid at position 12 is serine, and the amino acid at position 76 is valine, and the amino acid at position 80 is serine.
 14. The protein of claim 1, wherein the amino acid at position 76 is not cysteine, the amino acid at position 80 is not cysteine, and the amino acid at position 12 is not asparagine.
 15. The protein of claim 1, wherein the protein is a synthetic therapeutic protein.
 16. The protein of claim 1, wherein the protein increases electrical resistance in an in vitro transepithelial electrical resistance assay.
 17. A pharmaceutical composition, comprising: the protein of claim 1 and a pharmaceutically acceptable carrier.
 18. A method of treating a subject diagnosed with a gastrointestinal epithelial cell barrier function disorder, comprising: a. administering to a patient in need thereof a pharmaceutical composition, comprising: i. the therapeutic protein of claim 1; and ii. a pharmaceutically acceptable carrier.
 19. The method of claim 17, wherein the subject is diagnosed with a disease or disorder selected from the group consisting of: inflammatory bowel disease, ulcerative colitis, Crohn's disease, short bowel syndrome, GI mucositis, oral mucositis, chemotherapy-induced mucositis, radiation-induced mucositis, necrotizing enterocolitis, pouchitis, a metabolic disease, celiac disease, inflammatory bowel syndrome, and chemotherapy associated steatohepatitis (CASH). 